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Diagnosis of bovine brucellosis in Mosul city by indirect ELISA and conventional serological tests M. S. Rhaymah, K. A. AL-Saad,and O. KH. AL-Hankawe Department of Internal and Preventive Medicine, College of Veterinary Medicine, University of Mosul, Mosul, Iraq Abstract The study was conducted on 126 cattle (94 females and 32 males) of different ages (1->5 years) randomly selected from July 2007 to August 2008 in Mosul. Indirect ELISA test and other traditional tests (rose Bengal test, tube agglutination test and 2- mercapto-ethanol test) were used to determine the incidence of bovine brucellosis. The highest incidence of disease was recorded by Indirect ELISA, 23.01%, whereas it was 18.25%, 11.90% and 4.76% by rose Bengal, tube agglutination and 2Mercapto-ethanol tests, respectively. The highest incidence was in females in all serological tests and the highest incidence was in females at the age between 1-3 years whereas in males more than 3 years of age it was 23.07%. The results of tube agglutination test revealed the titer 1/40 occurred mostly compared with other titers. Six chronic cases were determined by 2mercapto-ethanol test. The degree of agreement of negative samples with rose Bengal test and indirect ELISA, tube agglutination, and 2- mercapto-ethanol tests was 94.17%, 100% and 100%, respectively, and by indirect ELISA with rose Bengal, tube agglutination and 2-mercapto-ethanol tests was 79.31%, 51.72% and 20.68%, respectively.

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30. Charanjeet MS, Katoch RC, Prasenject D, Rajinder K. Application of RBPT, SAT and Avidin-Biotin serum ELISA for detecting brucellosis among livestock in himachal Pradesh. Indian J comp Microbiol Immunol Infect Dis. 2004;25:15-18.

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32. Kolar J. Diagnosis and control of brucellosis in small ruminants. Prev Vet Med. 1984;2:215-225.

33. Quinn P J, Carter M E, Markey B, Carter G R. Clinical Veterinary Microbiology. 1st ed. London: Elservier; 1999.78-79p.

34. Mittal KR, Tizard I R, Barnum DA. Serological cross-reactions between brucella abortus and yersinia enterocoliticao: 9 Int. J Zoonoses. 1985;12:219-227.

35. Uzal FA, Carrrascoe A, Echaide S, Robles CA. Evaluation of an Indirect ELISA for the diagnosis of bovine brucellosis in Patagonia, Argentina. http://www.iaeaiorg/programmes/nafa/d3/puplic/uzal/indirect1.55pdf.

36. Gall D, Nielsen K. Serological diagnosis of bovine brucellosis: A review of test performance and cost comparison. Rev Sci tech Int Epiz. 2004;23:989-1002.

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Effect of immunization with protoscolices antigens of hydatid cysts on growth of Echinococcus granulosus in dogs M. A. Aljawady and M. N. Al-Shahery Department of Microbiology, College of Veterinary Medicine, University of Mousl, Mosul, Iraq Abstract The study was designed to evaluate the immune response of puppies, injected by different protein fractions extracted from protoscolieces, obtained from ovine hydatid cyst. Indirect heamagglutination revealed a remarkable increase in the antibody titers for the immunized groups (A1.5, A3, B1.5, and B3) before and after challenge when compared with the control.

Biological variations showed decline in numbers of adult Echinococcus granulosus in the immunized groups when compared with the control. Other variations proved dropping in numbers of the worms within the same immunized groups. Subsequent reductions of cestodes were reported which were 83.8%, 81.3%, 78.2% and 74.6% for the groups A3, A1.5, B3, and B1.5, respectively.

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2. Al-Khalidi NW. Investigation of the Immunity of Dogs to Echinococcus granulosus during the prepatent infection. [dissertation], Ohio State University; 1982:1-35p.

3. Zhang W, Li J, McManus DP. Concepts in Immunology and Diagnosis of Hydatid Disease. Clin Microbiol Rev. 2003;16(1):18-36.

4. Zhang WB, Zhang ZZ, Chi PS. Vaccination of dogs against Echinococcus granulosus using soluble antigens of protoscolieces.

Chin J Vet Sci Technol. 1999;29:21-22.

5. Smyth JD. In vitro culture of Echinococcus spp. Proc. 13th Int Cong Hydat Madrid 1985:84-95p.

6. Smyth JD, Barett NJ. Procedures for testing the viability of Human Hydatid cysts following surgical removal especially after chemotherapy. Trans Roy Soc Trop Med Hyg. 1980;74:649-652.

7. Taherkhani H, Rogan MT. General characterization of laminated layer of Echinococcus granulosus. Irn J Med Sci. 2000;25:3-4.

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# I (# D . [ 9. Robyte JF, White BJ. Gel permeation chromatography. II. Organic solvent protein precipitation. in: Biochemical Techniques, Theory and practice. Wadsworth Inc., Belmont, California, USA. 1987;88-268p.

10. Hudson L, Hay FC. Application of gel chromatography. In: practical immunology, 1st edition, p156. Blackwell Scientific publication Osney Mead, Oxford, London Edinburgh, U.K. North Balwyn, Victoria, Australia. 1976.

11. Eckert J, Schantz PM, Gasser RB, Torgerson PR, Bessonov AS, Movses-sian SO, Thakur A, Grimm F, Nikogossian MA. Geographic distribution and prevalence, p. 100-142. In: Eckert, J. Gemmell, M. A. Meslin, F.-X. and Pawlowski Z. S. (ed.), WHO/OIE manual on echinococcosis in humans and animals: a public health problem of global concern. World Organisation for Animal Health, Paris, France.

2001.

12. Alton GG. Recent development in vaccination against bovine brucellosis. Aust Vet J. 1978;54:551-557.

13. Dalsgaard K. Adjuvants. Vet Immunol Immunopathol. 1987;17:145152.

14. Jones WO, Emery DL, McClure SJ, Wagland BM. Changes in inflammatory mediators and larval inhibitory activity in intestinal contents and mucus during primary and challenge infections of sheep with Trichostrongylus colubriformis. Int J Parasitol. 1994;24:519-525.

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1. Andersen FL, Ouhelli H, Kachani M. Compendium on cystic echinococcosis in Africa and in Middle Eastern countries with Special Reference to Morocco. Brigham Young University print services, provo, Utah 84604,1997:54-85.

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Clinical study of foot and mouth disease in feedlot calves in Mosul region M. A. Abd- Alhameed and M. SH. Rhaymah Department of Internal and Preventive Medicine, College of Veterinary Medicine, University of Mosul, Mosul, Iraq Abstract The affected calves showed clinical signs including presence of vesicles at the bucal cavity and hoof (90.7%), fever (36%), salivation (32.9%), depression (20.5%), Anorexia (19.6%), loss of body weight (7.94%), lamness (15.9%), lesions at the muzzle (15.3%), presence of the lesions at the feet (6.0%), prostration (4.7%), diarrhea (3.3%), presence of the lesion at the mouth cavity (3.0%), paralysis of hind quarters (2.2%) and sudden death (1.6%). There was significant increase in the rectal temperature, respiration rates, and heart rates in the infected animals compared with control group. Recovered animals appeared some diseases including theileriosis (40.4%), tympany (22.8%), pneumonia (19.9%), foot abscess (14.7%), and babesiosis (2.2 %). The mortality rate varied between 3.4% to 27.9% in different herds and the mean was 3.3 % in all herds.

There was significant relationship between age of animals and the incidence of the disease.

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1. Donaldson AI. Foot and mouth disease: the principl features. Irish Vet J. 1987;41:325â&#x20AC;&#x201C;327.

2. Kitching RP. Clinical variation in foot and mouth disease: Cattles. Rev Sci Tech Int Epiz. 2002;21:499â&#x20AC;&#x201C;504.

3. Gibson CF, Donaldson AI. Exposure of sheep to natural aerosols of foot and mouth disease virus. Res Vet Sci. 1986;41:45â&#x20AC;&#x201C;49.

4. Radostitis OM, Gay CC, Bood DC, Hinchcliff KW. Veterinary Medicine: A textbook of the disease of cattle, sheep, goat, pigs, and horses. 9th ed. London: W.B. Saunders company; 2000.1059â&#x20AC;&#x201C;1066p.

5. Azab AS, Shahawy MI, Hassos EI. Detection of antibody to foot and mouth disease virus â&#x20AC;&#x201C;infection associated (VIA) antigen in cattle. Sera in Nineveah Iraq Zanco J.1987;5:2.

6. Al-Bana AS, Shony MO. Foot and mouth disease in Iraqi Native Gazella: Virus Isolation, serology and characterization.(masterâ&#x20AC;&#x2122;s thesis) Baghadad University; 1988.

9. Gailiunas P, Cottral GE. Presence and persistence of foot and mouth disease virus in bovine skin. J Bacteriol. 1966;91:2333â&#x20AC;&#x201C;238.

10. Kitching RP, Alexanderson S. Clinical variation in foot and mouth disease: Pigs Rev Sci Tech Int Epiz. 2002;21:513â&#x20AC;&#x201C;518.

11. Kitching RP, Hughes GJ. Clinical variation in foot and mouth disease: Sheep and goats. Rev Sci Tech Int Epiz. 2002;21:505â&#x20AC;&#x201C;512.

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2

Department of Microbiology, College of Veterinary Medicine, University of Mosul, Mosul, Iraq, Department of Microbiology, 3 Department of Animal Diseases, College of Veterinary Medicine, AL-Baath University, Syria

Abstract The study was designed to determine the types of Clostridium perfringens and their toxins in sheep with suspected enterotoxemia in Hama province, Syria. For this purpose, 89 bacterial isolates were isolated from 132 samples collected from dead and some slaughtered sheep with suspected enterotoxemia and diagnosed with classical diagnostic tests and by using enzyme-linked immunosorbent assay (ELISA) technique to determine the types and toxins of C. perfringens. These isolates appeared as G+ bulged rods with curved ends and were as positive to lecithinase, gelatin hydrolysis and sugar fermented, as where negative to catalase, oxidase, and indole. Based on the ELISA results all isolates were C. perfringens types A 84 isolate (94.38 %), D 3 (3.37 %), and C 2(2.25 %). Clostridium perfringens type A was the dominant type in cases of enterotoxemia in sheep in Hama province detected by ELISA test. The enterotoxaemia causes considerable economic loss to the sheep industry particularly in Hama province and generally in Syria. Therefore, it is recommended that a proper vaccination schedule against enterotoxemia should be implemented for sheep flocks in Hama province. These vaccines should provide adequate protective immunity against all C. perfringens types specially types A and D. Available online at http://www.vetmedmosul.org/ijvs

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1. Quinn PJ, Carter ME, Markey B, Carter GR. Clinical Veterinary Microbiology 6th ed. Edinburgh, New York: Mosby ; 2004. 191-208p.

2. Davies RH, Wray C. Seasonal variations in the isolation of Salmonella typhi- murium, Salmonella enteritidis, Bacillus cereus and Clostridium perfringens from environmental samples. Zentralbl Veterinarmed.

1996;43:119-127.

3. Songer JG. Clostridial enteric diseases of domestic animals. Clin Microbiol Rev. 1996;9:216-234.

4. Itodo AE, Adesiyun AA, Adekeye JO, Umoh JU. Toxintypes of Clostridium perfringens strains isolated from sheep, cattle and paddock soils in Nigeria.Vet Microbiol. 1986;12:93- 96.

5. El Idrissi AH, Ward GE. Evaluation of enzyme-linked immuno sorbent assay for diagnosis of Clostridium perfringens enterotoxemias. Vet Microbiol. 1992;31:389-396.

6. Miserez R, Frey J, Buogo C, Capaul S, Tontis A, Burnens A, Nicolet J. Detection of alpha- and-epsilon-toxigenic Clostridium perfringens type D in sheep and goats using a DNA amplification technique (PCR). Lett Appl Microbiol. 1998;26:382-386.

7. Greco G, Madio A, Buonavoglia D, Totaro M, Corrente M, Martella V, Buonavoglia C. Clostridium perfringens toxin-types in lambs and kids affected with gastroenteric pathologies in Italy. Vet J. 2005;170: 346-350.

8. Osturk G. Etiopathology of enterotoxaemia in small ruminants in ElazÄąg and surrounding cities. Turk J Vet Anim Sci. 1996;20:63-68.

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10. Duchesnes C, Granum PE, Menozzi MG, Peck M, Pelkonen S, Popoff M, Stackebrandt E and Titball R.EUROPEAN COMMISSIONEuropean Conce- rted Action QLK2-CT2001-01267: Clostridia in medical, veterinary and food microbiology Diagnosis and typing. Key Action 2 â&#x20AC;&#x201C; Control of infectious diseases EUR 21463 EN. 2006.

11. Henderson TG. The detection of Clostridium perfringens type D enterotoxin in the intestinal contents of animals by counter immunoelectrophoresis. N Z J Sci. 1984;27:423-426.

12. Naylor RD, Martin PK, Sharpe RT. Detection of Clostridium perfringens epsilon toxin by ELISA. Res Vet Sci. 1987;42:255-256.

13. McClane BA, Snyder JT. Development and preliminary evaluation of a slide latex agglutination assay for detection of Clostridium perfringens type A entero- toxin. J Immunol Methods. 1987;100:131136.

14. Martin PK, Naylor RD. A Latex agglutination test for the qualitative detec- tion of Clostridium perfringens epsilon toxin. Res Vet Sci. 1994;56:259-261.

15. Yoo HS, Lee SU, Park KY, Park YH. Molecular typing and epidemiological survey of prevalence of Clostridium perfringens types by multiplex PCR. J Clin Microbiol. 1997;35:228-232.

16. Kalender H, Ertas HB, Cetinkaya B, Muz A, Arslan N, KÄąlÄąc A. Typing of isolates of Clostridium perfringens from healthy and diseased sheep by multi- plex PCR. Vet Med-Czech. 2005;50:439442.

17. Nagahama M, Kobayashi K, Ochi S, Sakurai J. Enzyme-linked immuno- sorbent assay for rapid detection of toxins from Clostridium perfringens. FEMS Microbiol Lett. 1991;68(1):41-4.

18. Dela Rosa C, Hogue DE, Thonney ML. Vaccination schedules to raise antibody concentrations against epsilon-toxin of Clostridium perfringens in ewes and their triplet lambs. J Anim Sci. 1997;75:23282334.

19. Bernath S, Fabian K, Kadar I, Szita G, Barna T. Optimum time interval between the first vaccination and the booster of sheep for Clostridium perfrin- gens type D. Acta Vet Brno. 2004;73:473-475.

20. Anita CD, David CV, Paul SM, Ellis RP. Comparison of the odds of isolation,genotypes, and in vivo production of major toxins by clostridium perfringens obtained from the gastrointestinal tract of

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22. Gokce HI,, Genc O, Sozmen M, Gokce G. Determination of Clostridium perfringens Toxin-Types in Sheep with Suspected Enterotoxemia in Kars Province, Turkey. Turk J Vet Anim Sci. 2007; 31(5):355-360.

dairy cows with hemo-rrhagic bowel syndrome or left â&#x20AC;&#x201C;displaced abomasums.JAVMA 2005;227(1):132-138.

21. Quinn PJ, Markey BK, Carter ME, Donnelly WJC, Leonard FC. Veterinary Microbiology and Microbial Diseases. USA: Blackwell publishing Company; 2003. 84â&#x20AC;&#x201C;96p.

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Detection of anti brucella antibodies in lambs and goat kids using rose bengal test and indirect ELISA in Gugjeli - Ninavah province, Iraq S. A. Esmaeel, B. A. Mohmmed, S. D. Hassan and Kh. J. Hassain Department of Internal and Preventive Medicine, Collage of Veterinary Medicine, University of Mosul, Mosul, Iraq Abstract The object of the study was to detection of anti-brucella antibodies in lambs and goat kids by using Rose Bengal and indirect ELISA tests. Sera samples were collected from four flocks, including 135 samples (95 from lambs and 40 from goat kids). The sera examined by rose Bengal test showed negative result in all of flocks, in percentage was zero %. While the percentage of anti-brucella antibodies using indirect ELISA test in female and male lambs was (29.34 %, 10 %) respectively, with total percentage 25.3% and in female and male kids was (55 %, 0.0 %) respectively, with total percentage 27. 5%. The results showed incompatibility between rose Bengal test and indirect ELISA test with kappa value. The study concluded that indirect ELISA test can be considerable as a screening and diagnostic test instead of rose Bengal test for detection of antibrucella antibodies in lambs and kids.

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1. Refai M. Incidence and control of brucellosis in the Near East region.

Vet Microbial. 2002;20:81-110.

2. Radostits OM, Gay CC, Hincheliff KW, Constable PD. Veterinary Medicine: A text book of the diseases of cattle,horses, sheep, pigs and goats. 10 th ed,Saunders Elsevier,London. 2007;966-994.

3. Fich TA. Intracellular survival of Brucella defining the link with persistence. Vet Microbial. 2003;92:213-223.

4. Adams LG. The pathology of brucellosis reflects the outcome of the battle between the host genome and the brucella genome. Vet Microbial. 2002;90:1-4:553-561.

5. Alton GG. In: Nielson K, Duncan JR,eds. Animal brucellosis. Boca Raton, FL, CRC press; 1990. 379p.

6. Grillo MJ. Transmission of Brucella melitensis from sheep lambs. Vet Rec. 1997;140:32:602-605.

7. Alton GG, Jones LM, Angas RD, Verger JM. Techniques for the brucellosis laboratory INRA, Paris, 1988.

8. Martin SW. The evaluation of tests. Can J Com Med. 1997;41:19-25.

9. Ocholi RA, Kwaga JKP, Ajogi I, Bale JOO. Abortion due to Brucella abortus in sheep in Nigeria. Rev sci tech Off Int Epiz. 2005;3:973979.

10. Burgess GW, McDonald JW, Norris MJ. Epidemiological studies on ovine brucellosis in selected ram flocks. Australian Vet J. 2008;2(59): 45-47.

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15. Hussein AAA, Sayed ASM, El Feki MA. Seroepidemiological study on human brucellosis in Assiut Governorate. Egypt J Immumol 2005;12:49-56.

16. Gulaand ST, Khan A. Epidemiology and Epizootology of brucellosis: A review. Pakistan Vet J. 2007;27(3):145-151.

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Available online at http://www.vetmedmosul.org/ijvs

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6. Shaper AG, Pocock SJ, Walker M, Thompson AG. British regional heart study: Cardiovascular risk factors in middle-aged man in 24 towns. Br Med J. 1981;283:179-186.

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Bubulcusibis and Langhing gull larus atricullia. Arch Environ contan Toxicol. 1980;9:65-78.

11. Sakamoto J, Hashimoto K. Reproductive toxicity of arylamide and related compounds in mice: effect on fertility and sperm morphology.

Arch Toxicol. 1986;59:201-205.

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14. Teijon C, Jesus M, Socorr D, Juan A, Martin A, Lozano M, Bernardo M, Blanco M. Lead accumulation in rats at non acute doses and short periods of time: hepatic, renal and hematological effects.

Ecotoxicology and Environmental Restoration. 2000;3(1):36-41.

15. Queiros E, Waissmann W. Occupational exposure and effects on the male reproductive system. Cad Saude Publica. 2006;22(3):1-17.

16. Ahmad I, Sabir M, Yasin K. Study of the effects of lead on the poisoning on the testes in albino rate. Pakistan J Med Res, 2003;42(3): 160-168.

17. Ichikawa T, Oeda T, Ohmori H, Schilli WB. Reactive oxygen species influence the acrosome reaction but not acrosin activity in human spermatozoa. Int J Androl. 1999;22:37-42.

18. Batra N, Nehru B, Bansal M. Influence of lead and zinc on rat male reproduction at biochemical and histopathological levels. J Appl Toxicol. 2001;21(6):507-512.

19. Agency for Toxic Substance and Disease Registry ATSDR. Lead Toxicity. Physiologic Effects. 1999.1-9. www.atsdr.cdc.gov.

20. Hipler UC, Gornig M, Hipler B, Romer W, Schreiber G. Stimulation and scavestrogen-induced inhibition of reactive oxygen species generated by rat sertoli cells: Arch Androl. 2000;44:147-154.

21. Taupeau C, Poupon J, Treton D, Brosse A, Richard Y, Machelon V. Lead reduces messenger RNA and protein levels of cytochrome P450 aromatase and estrogen receptor β in human ovarian granulosa cells.

Biology of Reproduction (in press). 2003.

22. Sikka SC. Oxidative stress and role of antioxidants in normal & abnormal sperm function. Front Biosic, 1996;1:78-86.

23. Wohaieb SA, Godin DV. Starvation-related changes in free radical tissue defense mechanisms in the rat. Diabetes, 1987;36:169-173.

24. Jones R, Mann T, Sherins R. Peroxidative breakdown of phospholipids by human spermatozoa, spermicidal properties of fatty acid peroxides and protective action of seminal plasma. Fertil Steril, 1979;31:531-537.

25. Okasha S, Mousa S. Reproductive effects of lead acetate in adult male rats. J Egypt Ger Soc. 2001;27(A):113-129.

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Extensor and flexor digit synovial sheath, sac and synovial capsule in the distal part of the limbs in buffalos and camels and its relation of surgical interference S. AL-sadi1, G. Alosh2 and A. AL-Omari3 1

2

Department of Anatomy, College of Veterinary Medicine, University of Mosul, Mosul, Iraq, Department of Anatomy, 3 Department of Surgery, College of Veterinary Medicine, University of Al-baath, Hama, Syria

Abstract Sixty one samples of the distal parts of limbs were obtained from different ages of buffalo and camels of both sex to study the synovial structures to determine the suitable sites for injection of surgical interference. The result showed that extensor digit synovial sheath was extend between middle or distal part of metacarpal (metatarsal) to the extensor processes and this formed with synovial capsule dorsal pouches which serve in surgical interference. The flexor digit synovial sheath extended to palmar (planter) between distal extremity of metacarpal (metatarsal) to the middle of second phalanx in buffalo while in camel it extended to the proximal extremity of second phalanx, that sheath was formed with suspensory ligament and sessamoid bone palmar or planter pouches which were serve the surgical interference. Fourth synovial bursa observed situated dorsally

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between the extensor digit laterals tendon and capsule of fetlock joint, forms site of injection during surgical interference, while the other two synovial bursa were located to palmer (planter) between deep flexor tendon and distal sessamoid bone in buffalo while in camel these bursa were located between deep flexor tendon and cartilage of the second phalanx, these bursa were served for surgical interference. The synovial capsule which serve the surgical interference through digit cushion these were shown extended from the claw capsule. The result show that surgical interference was form six pouches in buffalo and eight pouches in camel, which formed by synovial structures and the tissue associated with them.

Available online at http://www.vetmedmosul.org/ijvs

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Diagnosis of foot and mouth disease by indirect competitive ELISA test in feedlot calves - in Mosul province M. A. Abd-Alhameed and M. Sh. Rhaymah Department of Internal and Preventive Medicine, College of Veterinary Medicine, University of Mosul, Mosul, Iraq Abstract Indirect competitive ELISA test used, for detection of non-structural protein (3ABC) in 460 serum samples of infected animals revealed that 421 samples were positive (91.5%) and 39 samples were negative (8.5%), with 91.5% sensitivity and 28.4% specifity. Blood picture revealed significant (P<0.05) increase in total leukocytic count, mean corpuscular volume, mean corpuscular hemoglobin and the percentage of granulocytes, and significant decrease in the total erythrocytic count and packed cell volume.

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5. Azab AS, Shahawy MI, Hassos EI. Detection of antibody to foot and mouth disease virus â&#x20AC;&#x201C;infection associated (VIA) antigen in cattle. Sera in Nineveah Iraq. Zanco. 1987;5:2.

6. Al-Bana AS, Shony MO. Foot and mouth disease in Iraqi Native Gazella: Virus Isolation, serology and characterization. (masterâ&#x20AC;&#x2122;s thesis). Baghdad: Microbiology, college of veterinary medicine.1988.

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# () 8. Kitching RP, Hughes GJ. Clinical variation in foot and mouth disease: Sheep and goats. Rev Sci Tech Off Int Epiz. 2002b;21:505 â&#x20AC;&#x201C; 512.

9. Radostitis OM, Gay CC, Bood DC, Hinchcliff KW. Veterinary Medicine. A textbook of the disease of cattle, sheep, pigs, and horses.

9th ed. London: W.B. Saunders company; 2000.1059â&#x20AC;&#x201C;1066p.

10. Donaldson AI, Hearps A, Alexandersen S. Evaluation of aportable "real time" PCR machine for FMD diagnosis. Vet Rec. 2001;149: 430.

11. Office International Des Epizooties (OIE). Foot and mouth disease chapter 2.1.1. in manual of standards for diagnostic test and vaccines.

4th ed. OIE, Paris. 2001;77â&#x20AC;&#x201C;92p.

12. Robiolo B, Seki C, Fondevilla N, Griger P, Scodeller E, Periolo O, Torre JL, Mattion N. Analysis of the immune response to FMDv structure and non-structural proteins in cattle in Argentina by the combined use pf liquid phase and 3ABC-ELISA tests. 2005.

13. Bergarman I, Malirat V, Neitzert E, Beck E, Panizzutti, N, Sanchez C. Improvement of a serodiagnostic strategy for foot and mouth disease virus surveillance in cattle under systemic vaccination: a combined system of indirect ELIZA-3ABC with an enzyme linked immuno electrotransfer blot assay. Arch Virol. 2000;145:473â&#x20AC;&#x201C;489.

14. Bergmann IE, Mello AP, Neitzert E, Beck E, Gomes I. Diagnosis of persistent apthovirus infection and its differentiation from vaccination response by use of enzyme linked immuno electrotransfer blot analysis with bioengineered non-structural viral antigens. Am J Vet Res.1989;54:825.

15. Thrusfield FM. Veterinary epidemiology. 7th ed. London: Butterworths; 1986.183p.

16. Diego DM, Brocchi E, Mackay D, Simone DF. The non â&#x20AC;&#x201C; structural poly protein 3ABC of foot and mouth disease as a diagnosis antigen in ELISA to differentiate infection from vaccinated cattle. Arch Virol.

1967;142:2021â&#x20AC;&#x201C;2033.

17. Clavijo A, Zhou EM, Hole K, Glic B, Kitching P. Development and of a biotinylated 3ABC recombinant protein in a solid â&#x20AC;&#x201C; phase competitive ELISA for the detection of antibodies against foot and mouth disease virus. J Virol Meth. 2004;120:217â&#x20AC;&#x201C;227.

18. Brousvoort BMDC, Sorensen KJ, Anderson J, Corteyn A, Tanya VN, Kitching RP, Morgan KL. Comparison of 3ABC Enzyme linked Immunosorbant Assay for diagnosis of multiple â&#x20AC;&#x201C; serotype foot and mouth disease in a cattle population in an area of endemicity. J. Clin.

Microbio. 2004;42:2108-2114.

19. Sorensen KJ, Madsen KG, Madsen ES, Salt JS, Niqindi J, Mackay DKJ. Differentiation of infection from vaccination in foot and mouth disease by the detection of antibodies to the non-structural proteins 3D, 3AB, and 3ABC in ELISA using antigen expressed I bacilovirus.

Arch Virol.1989;143:1461â&#x20AC;&#x201C;1476.

20. Coles EH. Veterinary Clinical Pathology. 4th ed. Philadelphia: W. B. Saunders Company; 1986. 10â&#x20AC;&#x201C;98p.

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1. Gokce G, Gokce HI, Gunes V, Erdogan HM, Citil, M. Alterations in some Haematological and Biochemical parameters in cattle suffering from foot and mouth disease. Turk Vet Sci. 2004;28:723-727.

2. James AD, Rushton J. The economics of foot and mouth disease. Rev Sci Tech Off Int Epiz. 2002;21:637 â&#x20AC;&#x201C; 644.

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4'. H = E 4. Kitching RP. Clinical variation in foot and mouth disease: Cattles. Res Sci Tech Int Epiz.2002a;21:499 â&#x20AC;&#x201C; 504.

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Evaluation the suitable level of standard concentrated ration for fattening growing Sharabi calves N. M. Abdullah1, A. K. Nasser2 and N. Y. Abou2 1

2

Department of Veterinary Public Health, College of Veterinary Medicine, University of Mosul, State of Board of Agriculture Research, Department of Agriculture Research, Nineveh, Mosul, Iraq

Abstract Three groups of growing Sharabi calves were used in this study to evaluate the best suitable level of concentrate ration.

Each group consisted of four calves between 8-12 months of age, with an average live body weight around 145 kg. They were fed for 90 days on concentrated standard ration according to NRC, 1984, at three different levels of 2, 3, 4 % of live body weight, respectively, consisted of 80:20 concentrate to roughage contained 14% crude protein, 72% TDN and 2.4 Mkcal/kg.

ME. The total gain and amount of consumed ration were measured. At the end of the trial, samples of rumen fluid were

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collected from all calves in order to estimate the activity of bacteria and micro organism. Results showed that the average amount of the daily ration consumed by the three groups of calves were 3.68, 6.91 and 8.12 kg/calves, respectively. There were significant differences (P<0.05) in average daily gain among the three groups of calves (311, 869 and 1000 gm, for the three respective treatments). Also the feed conversion ratio was improved for the second and third groups compared with the first group and they were 7.96, 8.12 and 11.8 kg. ration/kg live body weight gain, respectively. Ration levels showed no effect on rumen fluid pH but increased the time of methylene blue reduction (more than 15 mints) and sedimentation activity test (more than 20 minuets) than their normal standard levels. The cost to produce one kg live body weight gain was about the same for the second and third groups and it was 50% less than first group and they were 3184, 3248 and 4746 I.D., respectively. It was concluded that the fattening regime for growing Sharabi calves on concentrated ration given at 4% of live body weight for three months had more economic profit.

Available online at http://www.vetmedmosul.org/ijvs

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7. Rosenberger G. Clinical Examination of Cattle. 2 nd ed. Verlage Paul Parey, Berlin and Hamburg, Germany. 1977. 197-212p.

11. Arafat EA. A comparative study of the effect of different level of urea on performance of IRAQI local breed bull calves. [master's thesis], College of Agriculture and Forestry, University of Mosul; 1977.

12. Murphy TA, Loerch SC. Effects of restricted feeding growing steers on performance and carcass characteristics. Ohio Beef Cattle Research and Industry Report. 1993:93-1; 99-110p.

13. Hicks RB, Owens FN, Gill RD, Martin JJ, Strasia CA. Effects of controlled feed intake on performance and carcass characteristics of feedlot steers and heifers. J Anim Sci. 1993;68:233.

14. Plegge SD. Restricting intake of feedlot cattle, Oklohoma State Univ.

Stillwater. 1987.

15. Zinn RA, Owens FN. Influence of feed inake level on site of digestion in steers fed a high concentrate diet. J Anim Sci. 1983;56:471-475.

16. Brown MS, Ponce CH, Pulikanti R. Adaptation of beef cattle on highâ&#x20AC;&#x201C; concentrate diets: Performance and ruminal metabolism. J Anim Sci. 2006;84:E25- E33.

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The effect of substituting Nigella Sativa Meal as a source of protein in the rations of local rabbits on their productive performance and carcass traits N. M. Abdullah and A. A. AL-Kuhla Department of Veterinary Public Health, College of Veterinary Medicine, University of Mosul, mosul, Iraq Abstract Fifteen local male rabbits (6-8 weeks old) were allocated into three nutritional groups. The first group fed standard ration, 5 and 10% of the Nigella Sativa Meal (NSM) were added to the concentrated rations of the 2nd and 3rd groups replacing 36 and 72% of the soybean meal (SBM) protein respectively. The feeding period lasted for eight weeks. Feed consumption and body weight gain were recorded weekly. At the end of feeding period, all rabbits were slaughtered and carcass traits were studied.

No significant differences were found in total body weight gain and feed conversion rate (475, 502, 478 gm) and (4.8, 4.8, 4.9 kg ration/1 kg wt. gain). Feed cost per 1 kg body gain declined 16% in the 3rd group, which cost 2294 ID, compared with the 1st group (2717) and the 2nd group (2561 ID). No significant differences in all carcass traits were found. Substituting 72% of SBM protein by NSM protein in rabbit ration showed no negative effects on all productive parameters and carcass traits.

Available online at http://www.vetmedmosul.org/ijvs

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4. Khalifa MM. Nigella seeds oil meal as a protein supplement in broiler diets.(master's thesis), Face. Agric. Alexandria Univ. 1995.

5. EL-Nattat WS, EL-Kady RI. Effect of different medical plant seeds residues on the nutritional and reproductive performance of adult male rabbits. Int J Agric Biol. 2007;9:(3).

6. Zeweil HZ. Evaluation of substituting Nigella seed oil meal for soybean meal on the performance of growing and laying Japanese quail. Egypt. Poultry Sic. 1996;16:451-477.

7. Nasr AS, Attia AI, Rashwan AA, Abdine AMM. Growth performance of New Zealand White rabbits as affected by partial replacement of diet with Nigella sativa or soyaben meals. Egyptian J. Rabbits Sciences. 1996;6:129-141.

8. Zeweil HS, Ahmed MH, EL-Adawy MM, Zaki B. Evaluation of substituting Nigella seed meal as a source of protein for soybean meal in diets of New Zealand white rabbits, 9th World Rabbit congress, June 10-13, 2008, Veronaâ&#x20AC;&#x201C;Italy.

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Editor-in-Chief Prof. Dr. Fouad K. Mohammad (BVMS, MS, PhD) Editoria Secretary Dr. Nabeel A. Al-Hussary (BVMS, MSc, PhD) Executive Editorial Board Prof. Dr. Mohammed N. Al-Shahery (BVMS, DSc) Prof. Dr. Samih H. Arslan (BVMS, PhD) Prof. Dr. Fadwa K. Tawfeek (BVMS, MSc, PhD) Dr. Talal H. Hussein (BSc, MSc, PhD) Dr. Tariq S. Qubih (BVMS, MSc, PhD)

Vol. 24, No. 1, 2010

Iraqi Journal of Veterinary Sciences is printed and organized by: Prof. Dr. Muneer S. Al-Badrany Dr. D. M. Aziz Dr. M. O. Abdul-Majeed

Staff of the editorial office Dr. Muna H. I. Al-Zubaidy Dr. Nibal S. Michaeel Dr. Zeena Tariq

Papers published in the Iraqi Journal of Veterinary Sciences are available online as pdf files at http://www.vetmedmosul.org/ijvs

Iraqi Journal of Veterinary Sciences (Iraqi J. Vet. Sci.) ISSN 1607-3894 (Print), ISSN 2071-1255 (Online)

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References References to literature should be numbered (in brackets) consecutively in the text in the order in which they are first mentioned, and listed at the end of the manuscript. Titles of journals should be abbreviated according to the List of Journals Indexed for MEDLINE (ftp://nlmpubs.nlm.nih.gov/online/journals/ljiweb.pdf). Personal communications or unpublished data should be mentioned in the text, but not included in the list of references.

Examples Journal article: Bingham CM, Wilson PR, Davies AS. Real-time ultrasonography for pregnancy diagnosis and estimation of fetal age in farmed red deer. Vet Rec. 1990;126:102106.

Book: Wanamaker BP, Pettes CL. Applied pharmacology for the veterinary technician. 1st ed.

Philadelphia: W. B. Saunders Company; 1996. 372 p.

Chapter in a book: Chabala JC, Miller MW. Chemistry of antiprotozoal agents. In: Campbell WC, Rew RS, editors. Chemotherapy of parasitic diseases. New York: Plenum Press; 1986. p.

25-85.

Dissertation: Zhao C. Development of nanoelectrospray and application to protein research and drug discovery [dissertation]. Buffalo (NY): State University of New York at Buffalo; 2005.

276 p.

Master's thesis: Roguskie JM. The role of Pseudomonas aeruginosa 1244 pilin glycan in virulence [master's thesis]. Pittsburgh (PA): Duquesne University; 2005. 111 p.

Journal article on the Internet: Polgreen PM, Diekema DJ, Vandeberg J, et al. Risk factors for groin wound infection after femoral artery catheterization: a case-control study. Infect Control Hosp Epidemiol [Internet]. 2006 Jan [cited 2007 Jan 5];27(1):34-7. Available from: http://www.journals.uchicago.edu/ICHE/journal/issues/v27n1/2004069/2004069.web.pdf Internet Webpage: AMA: helping doctors help patients [Internet]. Chicago: American Medical Association; c1995-2007 [cited 2007 Feb 22]. Available from: http://www.ama-assn.org/.

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Papers published in the Iraqi Journal of Veterinary Sciences are available online as pdf files at http://www.vetmedmosul.org/ijvs

Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 Contents Research articles Detection of Brucella antigen in the aborted ovine fetal stomach contents using a modified ELISA test M. I. Al-Farwachi, B. A. Al- Badrani and Th. M. Al-Nima

Page

Effect of bone marrow and low power lasers on fracture healing with destruction of both periosteum and endosteum in rabbits M. G. Thanoon and S. M. Ibrahim

5

Concurrent aflatoxicosis and caecal coccidiosis in broilers A. M. Shareef

11

Molds and mycotoxins in poultry feeds from farms of potential mycotoxicosis A. M. Shareef

17

Prevalence of intestinal ciliate Buxtonella sulcata in cattle in Mosul T. M. Al-Saffar, E. G. Suliman, H. S. Al-Bakri

27

Clinical, haematological and biochemical studies of babesiosis in native goats in Mosul E. G. Sulaiman, S. H. Arslan, Q. T. Al-Obaidi, E. Daham

31

Effect of industrial product IMBO速 on immunosuppressed broilers vaccinated with Newcastle disease vaccine O. G. Mohammadamin and T. S. Qubih

37

Serodiagnosis of Johne's disease by indirect ELISA in ovine I. M. Ahmed

41

Effect of treating lactating rats with lead acetate and its interaction with vitamin E or C on neurobehavior, development and some biochemical parameters in their pups A. A. Hassan and H. M. Jassim

45

Use of saturated sodium chloride solution as a tissue fixative A. Al-Saraj

53

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Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (1-4)

Detection of Brucella antigen in the aborted ovine fetal stomach contents using a modified ELISA test M. I. Al-Farwachi*, B. A. Al- Badrani* and Th. M. Al-Nima** *Department of Internal and Preventive Medicine, College of Veterinary Medicine, University of Mosul, ** Veterinary Hospital in Mosul, Mosul, Iraq (Received November 17, 2008; Accepted July 13, 2009) Abstract This study was conducted on two flocks of sheep suffering from abortion in Mosul city, Iraq. The clinical findings in ewes were abortion during the 3-4 months of gestation period in the both flocks. The total percentage of abortion was 11.7 %, whereas stillbirth percentage was 4 %. Brucella spp. was isolated from four (33.3 %) of the 12 samples (stomach contents of the aborted fetuses). All culture â&#x20AC;&#x201C; positive samples had also positive with direct smears. By a modified enzyme-linked immunosorbent assay (ELISA), Brucella antigens were detected in the fetal stomach contents of 5 samples. The sensitivity and specificity of the modified ELISA were 100 % and 87.5 % respectively. The test had a good negative predictive value but only a moderate positive predictive value. Therefore, the test would be useful for confirming the existence of suspect disease.

Comparison of modified ELISA with bacterial isolation demonstrated a close agreement (Kappa value = 0.92). Of the 12 serum samples from aborted ewes, eight samples were positive with Rose Bengle test (66.7 %), more than 10 samples (83.3 %) were detected by indirect ELISA test.

Keywords: Ovine brucellosis, ELISA, Fetus, Stomach contents.

Available online at http://www.vetmedmosul.org/ijvs

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Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (1-4)

The following specimens were collected for diagnosis of the disease: 1. Bacterial isolation trials from stomach contents of the aborted fetuses were made on the modified Brucella agar medium supplemented with antibiotic (Himedia, India) and incubated at 37 oC in an atmosphere of 10 % CO2 and in air for 6 days and then Brucella colonies were identified and typed according to conventional methods (4). Suspected colonies were further identified and subcultured on Brucella spp. agar slants. We identified Brucella spp. isolates according to morphologic characteristics, microscopic appearance. Brucella spp. isolates were typed according to their CO2 requirement, H2S production, and growth in the presence of basic fuchsin and thionin at final concentrations of 20 µg/ml, as described (4, 5).

2. Direct smear were prepared from aborted fetal stomach contents, then stained with Koster stain and examined under light microscope (16).

3. Detection of the brucella antigen from aborted fetal stomach contents by modified ELISA: Apart of the samples was heated for 1 hr at 70 oC and centrifuged.

The precipitates were washed twice with phosphate buffered saline (PBS) and centrifuged. The precipitates were then used for ELISA. A hyperimmune antibrucella serum for using in the modified ELISA was prepared in sheep by four biweekly injections subcutaneously with live Brucella vaccine (Rev.1 strain of Brucella melitensis) produced in (CZ Veterinaria, Spain) with the standard dose of 1×109 colony forming units (CFU) (17).

Modified ELISA was done according to the general principals of immunoblotting (18). Brifly, 20µl of each of the samples and controls (BPS) were applied to nitrocellulose membrane (Amersham). The membrane was blocked in 5 % skim milk in PBS for 45 minutes.

Membrane was washed three times, 5 min each with PBS- T (PBS containing 0.05 % Tween 20), was placed in 1: 100 dilution of anti brucella serum in PBS- T and incubated at room temperature for 1 hr. The membranes were then incubated in conjugate goat antimouse IgG horse – radish peroxidase (Svanovir ®, Sweden) diluted 1: 1000 in PBS – T for 1 hr. The same washing procedure was repeated after incubation with the anti – sheep peroxidase conjugate (Svanovir ®, Sweden), followed by reaction with a chromogen – substrate solution consisting of 30 µl of 30 % H2O2 in 50 ml of PBS mixed with 30 mg of 4 – Chloro – 1 – naphtol in 10 ml of cold methanol. The results were determined by observation of violet – stained spots on the membrane.

4. Serum samples: 12 blood samples from all aborted ewes (12) were collected by venipuncture at 15 days after abortion. Rose Bengal (RBT), and indirect ELISA were used to detect antibodies against brucellosis. The Rose Bengal test was performed as described by (19). A

Introduction Ovine brucellosis is usually caused by Brucella melitensis, less frequently by B. abortus, and rarely by B. ovis (1,2). It is usually manifested by abortion, with excretion of the organisms in uterine discharges and in milk (2,3). Diagnosis depends on the isolation of Brucella spp. from aborted fetuses, milk or from tissues removed at postmortem examination (1). Alternatively, specific cellmediated or serological responses to Brucella antigens can be demonstrated (3,4).

The demonstration by modified acid-fast or immunospecific staining of organisms of Brucella morphology in abortion material or vaginal discharges provides presumptive evidence of brucellosis, especially if supported by serological tests (5). Whenever possible, the species and biovar should be isolated, and identified by phage lysis or oxidative metabolic tests, or both, and by cultural, biochemical and serological criteria (5-7). The recently developed polymerase chain reaction (PCR) and DNA-probe methods provide additional means of detection (8,9).

No serological test is appropriate for all epidemiological situations (6). The buffered Brucella antigen tests (Rose Bengal plate agglutination test and buffered plate agglutination test) are suitable for screening herds and individual animals (10). The reactivity of positive samples should be confirmed by the complement fixation test or by enzyme-linked immunosorbent assay (ELISA), both of which can also be used for both screening and confirmation (11,12). The serum agglutination test is inferior to other tests in specificity and sensitivity, and is not recommended if other procedures are available (12). Another immunological test was developed for detection of brucella antigen in the bovine fetal stomach contents as agar gel precipitation and counter – immunoelectrophoresis (13).

Counter – immunoelectrophoresis also was used for detection of brucella antigen in the stomach contents of aborted buffalo fetuses (14), while coagglutination test was used for detection of Brucella antigens in aborted ovine fetal stomach contents (15). The aim of the study to describe a simple and rapid method (Modified ELISA) for detection of Brucella antigen in aborted ovine fetal stomach contents.

Materials and methods The study was conducted in April 2008 on two flocks of sheep in the Mosul city (Badoosh and Zumar) covering 120 and 52 sheep for each respectively. Complete examination was performed to all aborted animals and 12 were subjected to thorough postmortem examination. The two flocks were not vaccinated against brucellosis.

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Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (1-4)

commercially available indirect ELISA (Brucella – Ab IELISA kit Svanovir ®, Sweden) was used to detect specific antibodies for Brucella spp. according to manufacturer´s instruction.

Statistical analysis: The sensitivity, specificity, positive and negative predictive values were calculated. The Kappa value was used to evaluate the correlation between modified ELISA and bacterial isolation (20,21)

From the results, it appeared that the modified ELISA has a high sensitivity (100 %), but only moderate specificity (87.5 %). The test also has a good negative predictive value(100 %) and moderate positive predictive value (80 %). Therefore, the test is would be useful for confirming the existence of suspected disease. Comparison of modified ELISA with bacterial isolation demonstrated a close agreement (Kappa value = 0.92). Of the 12 serum samples from aborted ewes, eight samples were positive with Rose Bengle test (66.7 %), more than 10 samples (83.3%) were detected by indirect ELISA test.

Results Clinical examination showed abortion occurred during the 3-4 months of gestation period in the both flocks. Table 1 illustrate the number of aborted ewes and average of their gestation period in months. The total percentage of abortion was 11.7 %, while stillbirth percentage was 4 %.Brucella spp. was isolated from 4 (33.3 %) of the 12 samples (stomach contents of the aborted fetuses). All culture – positive samples had also positive results with direct smears (stained with Koster stain). By modified ELISA, Brucella antigens were detected in the fetal stomach contents of 5 samples (42.0 %). Other culture – negative samples were also negative with modified ELISA test (Table 2).

Discussion Brucellosis is widely regarded as an extremely insidious disease, demanding the most exhaustive care in diagnosis and many cases are more difficult to diagnosis than others, and a few are extremely difficult to pick up, and could easily be missed altogether (1,2). In this study, Brucella spp. was isolated from 4 (33.3 %) of the 12 samples (stomach contents of the aborted fetuses). All culture – positive samples were also positive with direct smears (stained with Koster stain). By modified ELISA, Brucella antigens were detected in the fetal stomach contents of 5 samples (42.0 %). Other culture – negative samples were also negative with modified ELISA test. According to the results of this study modified ELISA is able to detect Brucella antigens in fetal stomach content because the modified ELISA has a high sensitivity (100 % = false negative rate of 0%), but only moderate specificity (87.5 % = false positive rate of 12.5%). The test also has a good negative predictive value (proportion of modified ELISA negative animals which do not have disease = 100 %) and moderate positive predictive value (proportion of modified ELISA positive animals which have the disease = 80 %) (20). Therefore, the test would be useful for confirming the existence of suspected disease because the test has a few false positive rate and moderate positive predictive value (21).

Comparsion of modified ELISA with bacterial isolation demonstrated a close agreement (Kappa value = 0.92).The Kappa test can be used to measure the level of agreement beyond that which may be obtained by chance. The Kappa lies within a range between -1 and +1 (20). (20), (21) were noted that the Kappa value gives no which of the tests is better and that a good agreement may indicate that both test are equally good or equally bad.

Table 1: Numbers of aborted animals and average of their gestation period in months.

Regions

Total lambing

Abortion No (%)

Stillbirth No. (%)

Badoosh Zumar Total

120 52 172

8 (9.6 %) 12 (6.3 %) 20 (11.7%)

3 (3.6 %) 4 (2.0 %) 7 (4.0 %)

Gestation period (Months) 3-4 3 - 3.5 3-4

Table 2: Results of the tests used for detection of brucella antigens in stomach contents of the aborted fetuses.

Bacterial Direct culture smears 1 2 + + 3 4 + + 5 6 + + 7 8 9 10 + + 11 12 Total 4 (33.0 %) 4 (33.0 %) (-) negative results, (+) positive results.

Samples

Modified ELISA + + + + + 5 (42.0 %)

Acknowledgment This study was supported by the College of Veterinary Medicine, University of Mosul, Mosul, Iraq.

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References 12.

1. Radostits OM, Gay CC, Hinchcliff KW, Constable PD. Veterinary Medicine: A textbook of the diseases of cattle, horses, sheep, pigs and goats. 10th ed. London: Saunders Elsevier; 2007. 966-994p.

2. Cutler SJ, Whatmore AM, Commander NJ. Brucellosis- new aspects of an old disease. J Applied Microbiol. 2005;98:1270-1281.

3. Garin-Bastuji B, Blasco JM, Marin C. The diagnosis of brucellosis in sheep and goats, old and new tools. Small Rum Res. 2006;62:63-70.

4. Forbes BA, Sahm DF, Weissfeld AS. Diagnostic Microbiology. 11th ed. USA: Elsevier; 2002. 196-201p.

5. Songer JG, Post KW. Veterinary microbiology. Bacterial and fungal agents of Animal Disease. Missouri: Saunders Elsevier; 2005. 200206p.

6. Nielsen K. Diagnosis of Brucellosis by Serology. Vet Microbiol. 2002; 90:447-459.

7. Aggad H. Serological studies of animal brucellosis in Algeria. Assiut Vet Med J. 2003;49(98):121-130.

8. Fekete A, Bantle A, Halling SM. Detection of Brucella by polymerase chain reaction in bovine fetal and maternal tissues. J Vet Diag Invest.

1992;11:79-83.

9. Gallien P, Dorn C, Alban G, Staak C, Protz D. Brucella species in organs of naturally infected cattle by polymerase chain reaction. Vet Rec. 1998;142:512-514.

10. Baum M, Zamir O, Bergman-Rios R, Katz E, Beider Z, Cohen A, Banai, M. Comparative evaluation of microagglutination test and serum agglutination test as supplementary diagnostic methods for brucellosis. J Clinic Microbiol. 1995;33(8):2166-2170.

11. Blasco JM, Garin-Bastuji B, Marin CM, Gerbier G, Fanlo J, Jimenez de Bagues MP, Cau C. Efficacy of different rosebengal and

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complement fixation antigens for the diagnosis of Brucella melitensis in sheep and goats. Vet Rec. 1994;134:415-420 Minas A, Stournara A, Christodoulopoulos G, Katsoulos PD. Validation of a competitive ELISA for diagnosis of Brucella melitensis infection in sheep and goats. The Vet J. 2007;111:102-105.

Chand P, Gupta RK, Khanna RN, Sadana IR. Detection of Brucella antigen in bovine fetal stomach contents by agar gel precipitation and counter immunoelectrophoresis. Res Vet Sci. 1987;43(1):132-136.

Chand P, Khanna RN, Sadana JR. Counter â&#x20AC;&#x201C; immunoelectrophoresis for the detection of Brucella antigen and antibodies in the diagnosis of brucellosis in buffaloes. J Appl Bacteriol. 1988;64(5):445-449.

Erganis O, Kaya O, Hadimli H, Guler L. Rapid diagnosis of ovine Brucella,Campylobacter and Salmonella infections from fetal stomach contents by coagglutination test. Small Rum Res. 2002;45:123-127.

Coles EH. Veterinary Clinical Pathology. 4th ed. Philadelphia: W.B. Saunders Company; 1986. 43-79p.

Little SF, Ivins BE, Fellows PF, Friedlander AM. Passive protection by polyclonal antibodies against Bacillus anthracis infection in guinea pigs. Infect Immunol.1997;65:5171â&#x20AC;&#x201C;5175.

Golds RA, Kinde TJ, Osborne BA, Kaby J. Enzyme linked immunosorbent assay: In immunology 5th ed. Philadelphia: W.B. Saunders Company; 2003. 43-79p.

Alton GG, Jones LM Angus RD, Verger JM. Techniques for the brucellosis laboratory. Paris: INRA;1988.

Thrusfield M. Veterinary Epidemiology 2nd ed. Oxford: Blackwell Science Ltd; 1995. 95- 112p.

Pfeffer D. Veterinary Epidemiology. An Introduction. NewZealand: Institute of Veterinary, Animal and Biomedical Sciences.Massey University; 2000. 32-34p.

Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (5-9)

Effect of bone marrow and low power lasers on fracture healing with destruction of both periosteum and endosteum in rabbits M. G. Thanoon and S. M. Ibrahim Department of Veterinary Surgery and Theriogenology, College of Veterinary Medicine, University of Mosul, Mosul, Iraq (Received December 18, 2008; Accepted July 13, 2009) Abstract Ten mature rabbits of local breed were used in this study; weighing between 1.5 to 1.75 kg and aged about 1â&#x20AC;&#x201C;2 years.

These animals were divided into two equal groups; in group A destruction of both periosteum and endosteum was done one centimeter from each side of mid-shaft femoral bone fracture, then sufficient amount of autogenously bone marrow was injected directly at the fracture site after immobilization by intramedullary pin. In group B a similar procedure was achieved as in group A, but in additional to that He-Ne infrared laser therapy was used for several sessions. The result of radiological findings indicated that, the fracture healing occurred within group B at fifteen weeks, whereas in group A the healing occurred at eighteen weeks after operation. The implantation of autologous bone marrow enhanced the fracture healing, whereas using of combinations of autologous bone marrow and He-Ne infrared laser therapy hastened the healing.

Keywords: Fracture, Bone marrow, Laser, Rabbit.

Available online at http://www.vetmedmosul.org/ijvs

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Autologous bone marrow is a safe, simple and reliable method for treating delayed and nonunion (6-8). Low energy laser irradiation has positive effects on bone fracture healing. The mechanisms by which low-energy laser irradiation affect on bone healing still not clear (9). He-Ne low-level energy treatment accelerated the deposition of

Introduction Studies suggested that, the periosteum was more essential than endosteum in callus formation. If there is destruction of both periosteum and endosteum the results will be absence of bone formation around the destructed area (1,2). Bone marrow stem cells residing in the bone

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Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (5-9)

bone matrix and increases vascularization after seven days of irradiation (10). Direct irradiation of the whole injury with He-Ne laser on days five, six post-injury altered the osteoblast and osteoclast cell population (11,12). Studies on animals were performed on the effect of low-level laser of fracture healing indicated that, the laser enhanced healing (13-15). The aim of this study was to investigate the effect of bone marrow and low-level laser energy on healing of femoral fracture after destruction of both periosteum and endosteum in rabbits.

Results The results of this study showed that mild inflammation at the site of operation during the first few days after operation, theses signs represented by; pain, red, hot, swelling, but subsided quickly during fifth days in group A and fourth days in group B. The callus formation was tested clinically by stress of the fracture site by finger palpation, at the end of 2nd week in group A and at the end of 3rd week on B. It has noticed that the callus formation around the fractured area in group A larger than in group B. The callus disappeared clinically at eight and six weeks in group A and B respectively. The radiographical finding revealed that, the healing occurred at the eighteenth and fifteenth week in group A and B respectively, which characterized by the invisibility of fracture line and the bone taken about the normal shape. The periods of fracture healing are summarized in (Table 1).

Materials and methods Ten mature rabbits of both sex of local breed aged 1-2 years and weighing between 1.5-1.75 kg. All rabbits tolerated on the same manner of condition and housing along the periods of study. The experimental design divided into two equal series; Group A: five animals underwent transverse fracture at the mid-shaft of femur bone then about one centimeter of both periosteum and endosteum has been destroyed and treated with sufficient amount of bone marrow, Group B: five animals underwent the same conditions of group A in additional to that irradiated with He-Ne infrared laser for several sessions.

Surgical procedure was performed under general anesthesia by using protocol of anesthesia include atropine sulphate 1mg/kg b.w. intramuscularly as a premedication after fifteen minutes later a mixture of Xylazine and Ketamine hydrochloride given intramuscularly at a dose of 5 and 20 mg/kg b.w., respectively. The surgical site was prepared under aseptic technique. Skin incised directly at the mid-shaft of femoral bone to expose the bone and osteotomy was performed, about one centimeter of periosteum and endosteum were destroyed from both sides of fractured end with bistoury scalpel. The fractured ends fixed by stainless steel 2.4Ď&#x2020;Ă&#x2014;120 mm intramedullary pinning. Muscles sutured by simple continuous using catgut NO. 2/0, then sufficient amount of bone marrow which aspirated from femoral head of other side (using needle gauge 18) was injected at the site of fracture in-between the stitches, after that, suture the fascia and skin. While in group B in additional to that, animals were exposed to radiation with He-Ne infrared lasers (the He-Ne wave length: 632.8 nanometers, frequency: 50-60 Hz were the wave length and frequency of infrared are 904 nanometers, 700-1200 Hz respectively) applied at the fractured site, in a series of eight sessions (1-3 , 5, 7, 9, 11 and 13 days) after operation, the total dose of He-Ne infrared lasers for each animal was 3.6 joule/cm2.

Post-operative care by using penicillin-streptomycin at a dose 10000 I.U, 10 mg/kg b.w. injected intramuscularly for four days. Clinical examination and weekly radiological studies were performed for the fracture site to determine the stage of fracture healing.

Discussion The periosteum, endosteum and bone marrow provided cells that proliferate and differentiate into osteoblasts, chondroblasts and fibroblasts, which contribute to new bone formation (2,3). It was demonstrated that autologous bone marrow contains mesenchymal stem cells that are able to form bone, cartilage and enhancement of the osteogenesis in fracture healing (5,6).

In our study the destruction of both periosteum and endosteum at the fracture site and injection of sufficient amount of autologous bone marrow leaded to healing at about eighteenth week. This observation was confirmed by other authors (5,7). Who said that, the autologous bone marrow enhanced of bone healing. On the other hand this result agreed with other authors (1,2). Who recorded that, the periosteum and endosteum are important in fracture healing, which when both removed at the fracture area resulting in absence of callus formation.

Low level laser therapy can accelerate the bone regeneration. This was appeared by altered osteoblast activity at the fracture site as reflected by alkaline phosphatase activity (11). Laser irradiation also caused a significant increase in calcium accumulation at the fracture site (13). In this study, the results confirm these facts, which revealed that, the laser therapy enhanced the fracture healing in-group B when compared with group A. The destruction of both periosteum and endosteum at the fracture site then treated with a combination of laser and autologous bone marrow as in group B, which exhibited better results than using autologous bone marrow alone by other authors (6-8). We believed that this combination of autologous bone marrow and He-Ne infrared laser therapy may be not mentioned in literature.

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Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (5-9)

Table 1: Radiographic findings of group A and group B. Week 1 2 3 5 8

10 12 15 18

Group A Slight proliferation of periosteum at a distance from fracture site. Clear fracture line.

External callus become active and migrated in to the fracture site. Clear fracture line.

Increase internal callus and partially bridged fracture site. Clear fracture line.

Thick external callus bridged the fracture site. Still clear fracture line (fig. 1).

External callus began to absorb. Still visible fracture line (fig. 2).

Thin external callus around the fracture site. Invisible fracture line.

The bone began to taken about the normal shape. Thin external callus still around the fracture site (fig. 5).

Still visible external callus. Bone almost taken the normal shape.

Slight invisible callus. Bone may be taken about the normal shape.

Group B Slight proliferation of periosteum at a distance from fracture site. Clear fracture line.

Slight increase of external callus around the fracture site.

Clear fracture line.

External callus around the fracture site. Visible fracture line.

Thin external callus bridged fracture site. Still visible fracture line.

Good alignment of the cortex at the fracture site. Bone almost taken about the normal shape. Disappearance of fracture line (fig. 3).

Thin external callus around the fracture site. The bone taken about the normal shape (fig. 4).

The bone taken the normal shape. Still appear very thin external callus around the fracture site (fig. 6).

The bone is normal in shape. Still very thin external callus around the fractured area (fig. 7).

Fig. 1: Radiographic picture shows thick external callus bridged the fracture line. Still clear fracture line, five weeks after operation in group A.

Fig. 3: Radiographic picture shows bone almost taken the normal shape. Invisible fracture line, eight weeks after operation in group B.

Fig. 2: Radiographic picture shows external callus began to absorb. Still visible fracture line, eight weeks after operation in group A.

Fig. 4: Radiographic picture shows thin external callus around the fracture site. The bone taken about normal shape, ten weeks after operation in group B.

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Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (5-9)

this may be due to the He-Ne infrared lasers causes high activity of the source of osteoblasts and mesenchymal stem cells of autologous bone marrow for direct formation of bony material to fill the gap of fracture line instead of cartilage or fibrocartilage tissue formation as seen in common stages of fracture healing. The bone return to the normal feature approximately in eighteenth week in group A and fifteenth week in group B, this variation of period between these both groups may be due to the action of combination of autologous bone marrow and He-Ne infrared laser which accelerate the osteoblasts and osteoclasts activity to deposition and resorption of bone matrix.

In conclusion of this study which revealed that, the using of autologous bone marrow was enhanced the fracture healing. While using of combinations of autologous bone marrow and He-Ne infrared laser therapy caused more hastened healing.

Fig. 5: Radiographic picture shows the bone began to taken about the normal shape. Thin external callus around the fracture site, twelve weeks after operation in group A.

Acknowledgement This study was supported by the College of Veterinary Medicine, University of Mosul.

References 1. Kojimoto H, Yasui N, Goto T, Matsuda S, Shimomura Y. Bone lengthening in rabbits by callus distraction. The role of periosteum and endosteum. J Bone Joint Surg Br. 1988;70(4):543-549.

2. Guichet JM, Braillon P, Bodenreider O, Lascombes P. Periosteum and bone marrow in bone lengthening: a DEXA quantitative evaluation in rabbits. Acta Orthop Scand. 1998;69(5):527-531.

3. Reynders P. Intra-osseous injection of concentrated autologous bone marrow in 62 cases of delayed union. Folia Traumat Lovaniensia.

2003;108-116.

4. Porter SE, Hanley EN. The musculoskeletal effect of smoking. J AM Acad Orthop Surg. 2001;9(1):9-17.

5. Srouji S, Livne E. Bone marrow stem cells and biological scaffold for bone repair in aging and disease. Mech Ageing Dev. 2005;126(2):281287.

6. Sim R, Liang TS, Tay BK. Autologous marrow injection in the treatment of delayed and non-union in long bones. Singapor Med J. 1993;34(5):412-417.

7. Liang YT, Zhang BX, Lu SB. Experimental study and clinical application on osteogenesis of percutaneous autogenous bone marrow grafting in bone defects. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 1999;13(3):148-151.

8. Connolly JF, Guse R, Tiedeman J, Dehne R. Autologous marrow injection as a substitute for operative grafting of tibial nonunions. Clin Orthop Relat Res. 1991;(266):259-270.

9. Ninomiya T, Miyamoto Y, Ito T, Yamashita A, Wakita M, Nishisaka T. High-intensity pulsed laser irradiation accelerates bone formation in metaphyseal trabecular bone in rat femur. J Bone Miner Metab.

2003;21(2):67-73.

10. Garavello I, Baranauskas V, da Cruz-Hofling MA. The effects of low laser irradiation on angiogenesis in injured rat tibiae. Histol Histopathol. 2004;19(1):43-48.

11. Yaakobi T, Maltz L, Oron U. Promotion of bone repair in the cortical bone of the tibia in rats by low energy laser (He-Ne) irradiation. Calcif Tissue Int. 1996;59(4):297-300.

Fig. 6: Radiographic picture shows the bone taken the normal shape. Still appear very thin external callus around the fracture site, twelve weeks after operation in group B.

Fig. 7: Radiographic picture shows normal shape of bone.

Very thin external callus around the fracture area, fifteen weeks after operation in group B. The radiographic findings revealed that, the healing of the fractured bone is noticed in eighteenth, fifteenth week in group A and B respectively. These findings may be due to the He-Ne infrared laser therapy enhancement of osteogenesis and hasten the healing. This result was agreed with other authors (11,13). Who said that, the low level laser therapy can accelerate the osteogenesis. The external callus was larger in animals of group A than in group B,

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Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (5-9)

12. Barushka O, Yaakobi T, Oron U. Effect of low-energy laser (He-Ne) irradiation on the process of bone repair in the rat tibia. Bone J. 1995;16(1):47-55.

13. Luger EJ, Rochkind S, Wollman Y, Kogan G, Dekel S. Effect of lowpower laser irradiation on the mechanical properties of bone fracture healing in rats. Lasers Surg Med. 1998;22(2):97-102.

14. Guzzardella GA, Fini M, Torricelli P, Giavaresi G, Giardino R. Laser stimulation on bone defect healing: an in vitro study. Lasers Med Sci. 2002;17(3):216-220.

15. Khadra M, Kasem N, Haanaes HR, Ellingsen JE, Lyngstadaas SP. Enhancement of bone formation in rat calvarial bone defects using low-level laser therapy. Oral Surg Oral Med Oral Pathol Oral Radijol Endod. 2004;97(6):693-700.

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Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (11-16)

Concurrent aflatoxicosis and caecal coccidiosis in broilers A. M. Shareef Department of Veterinary Public Health, College of Veterinary Medicine, University of Mosul, Mosul, Iraq (Received January 27, 2008; Accepted July 13, 2009) Abstract A farm of two broiler flocks fed from one day old to 56 days of age a diet naturally contaminated with high level of Aflatoxin (AF), were suffered from caecal coccidiosis at 35 days of age Aflatoxin levels in feed commodities and mixed feed were determined using ELISA test. Results of ELISA showed that the average level of corn, soybean and mixed feeds contamination with aflatoxin were 1915, ppb, 229 ppb and 860 ppb respectively. Diagnosis of caecal coccidiosis was confirmed by clinical signs, post-mortem findings, fecal oocyst excretion per gram, oocyt size, morphological characteristic of eggs, lesion score and schizonts size. The significance of AF as predisposing factor to coccidiosis infection was discussed.

Keywords: Coccidiosis, Aflatoxin, Broilers.

Available online at http://www.vetmedmosul.org/ijvs

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2 3 1 N # ,O P 1 2 ! , maize, sorghum grain, barley, wheat, rice meal, cottonseed meal, groundnuts and other legumes. Most are relatively stable compounds and are not destroyed by processing of feed and may even be concentrated in screenings. Naturally occurring toxins present a different problems (5,6).

Mycotoxins cannot be avoided entirely or eliminated from animal feeds because the molds that create them occur naturally in grains and other food commodities Aflatoxins B1, B2, G1, and G2 are mycotoxins that may be produced by three moulds of the Aspergillus species: A. flavus, parasiticus and A. nomius (7) Aflatoxin B1 is an immunosuppressive mycotoxin for cell-mediated immunity

Introduction Poultry aflatoxicosis was first reported when 100000 turkey poults in the United Kingdom in 1960 died following the ingestion of poultry feed containing Brazilian groundnut cake which led to the discovery of a group of compounds now called the aflatoxins (1). Soon afterwards, however, aflatoxicosis became a worldwide problem (2-4).

Mycotoxins are secondary metabolites produced by fungi of various genera when they grow on agricultural products before or after harvest or during transportation or storage.

Mycotoxins are regularly found in feed ingredients such as

11

Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (11-16)

rate of 26% with signs of reduction in feed consumption, depression, ruffled feathers, closed eyes, stunted growth, and profuse bloody diarrhea. These birds were fed a commercial diet, with 22% protein and 3060 kcal/kg metabolizable energy, without coccidiostatic or Aflatoxin binder additives.

(CMI), humoral immunity, suppression of peritoneal macrophage phagocytosis and the primary splenic antibody response (8-11). Aflatoxin has been reported to increase the severity of avian infectious diseases such as salmonellosis (12), crop mycosis (13), aspergillosis (14), and coccidiosis (15).

Avian coccidiosis is the major parasitic disease of poultry with a substantial economic burden on the cost the industry (16). In-feed medication for prevention and treatment contributes a major portion of these losses in addition to mortality, malabsorption, inefficient feed utilization and impaired growth rate in broilers, and a temporary reduction of egg production in layers. The pathogenicity of coccidia depends largely on the successful replication of developing parasites inside the host.

Theoretical estimates indicate that a single oocyst of a virulent species such as E. tenella could yield 2,520,000 invasive parasites after the 2nd merogony stage (17). The profuse bleeding in the ceca is a characteristic feature of E. tenella infection due to its extensive destruction of the mucosa with histological lesions (18). In general, young animals are more susceptible to coccidiosis and more readily display signs of disease, whereas older chickens are relatively resistant to infection (19). Young animals which recover from coccidiosis may later be able to partly compensate for the loss of body growth, but their growth potential remains severely compromised. Because the life cycle of Eimeria parasites is complex and comprised of intracellular, extracellular, asexual, and sexual stages, host immune responses are quite diverse and complex. After invasion of the host intestine, Eimeria elicit both nonspecific and specific immune responses which involve many facets of cellular and humoral immunity (20,21) Caecal coccidiosis of broilers has been extensively studied (22), and as the worlds poultry industry has developed the disease has continued to be of major economic importance (23). Interactions occur between chicken caecal coccidiosis and other diseases, caused by various pathogens (24).

Among those other diseases, aflatoxin intercurrent with coccidiosis are an increasing health risk to poultry and poultry coccidiosis due to primary exposure to aflatoxin had been reported previously (25-30). A natural broiler aflatoxicosis or broiler caecal coccidiosis had been reported in mosul province (4, 31).

The natural concurrence of these both cases in broilers is what we are tried to describe in a farm of two broiler flocks, in Mosul province, Iraq.

Pathological examination: Forty chicks from the two flocks were necropsed for pathological examination. These birds were scored for cecal lesions as devised by (32) as follows; Score 0: no gross lesions; Score1: very few scattered petichiae on the caecal wall with normal caecal contents; Score 2: lesions are more numerous, blood in content, wall is somewhat thickened, normal caecal content; Score 3: large amounts of blood or caecal cores, caecal wall greatly thickened, little if any fecal content in ceca; Score 4: cecum is greatly distended, blood or caseous core, no fecal debris, dead birds; +4.

Ten of these dead chicks were subjected to caecal mucosal scraping for schizont determination. Oocyst size and shape were determined from about 50 predominant oocysts. A concentration McMaster technique has been used for calculation of average fecal oocyst excretion per gram (33).

Feed sampling: Twenty five feed samples (each 1 kg) were taken from the broiler farm feed store, five samples of corn, barley, wheat, soybean and mixed feed according to (34).

Aflatoxin assay: The levels of aflatoxin contamination of feed samples were determined by the method of direct competitive enzyme-linked immunoassay using Neogen extraction kit (Neogen Corporation). Results of the yield optical densities of the controls and samples were obtained by using computerized Neogen Verotex Software program version 2.0.16 (Neogen Corporation).

Results The most prominent lesions seen in 80 necropsid birds in both flocks were summarized in (Table 1), in which pathological changes were mainly seen in the liver and two caecai. Those in the liver were characterized by creasy, yellow, ocher discoloration, with scattered areas of sub capsular hemorrhage. The caecal lesions were characterized by accumulation with varying quantities of blood and caseous necrotic materials (Figure 1). From table 1, it is evident that 5% of necropsed broilers in both flocks show (+) intensity of liver discoloration. More liver discoloration intensity (++) were recognized in both flocks with 22.5% in flock No.1, and 24% in flock No.2. Similar percentage of liver discoloration changes intensity (+++) were noticed in

Materials and methods Chicks and diet: A broiler farm, in Al-Hamdanyea / Mosul province, reared on letter 8600 and 9650 broilers in two separate house, had been claimed at 35 days of age from mortality

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Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (11-16)

both flocks. Higher percentage (37.5%) of liver intensity (++++) was recorded in flock No. 1, than that of 35% in flock No. 2. The intensity of sub capsular hemorrhage in livers of necropsed broiler chicks in both flocks, showed that only 2.5% of the necropsed birds in flock No. 1 with sub capsular hemorrhage of (+) was recorded, in comparison of 10% in flock No. 2. More sub capsular hemorrhage intensity (++) was noticed in flock No. 1 (27.5%), compared with 25% in flock No. 2. Still more intensity of (+++) in flock No. 1 of 35% was recorded, compared with 30% in the other flock. The same picture was continued with the highest sub capsular hemorrhage intensity of (++++) was also recorded in flock No. 1 with 37.5%, while it was 35% in flock No. 2.

N0.1was about, they were 28 and 16% respectively. Two percent of score 2 was recorded in both broiler flocks.

Relatively similar percentage for both broiler flocks 1 and 2, and they were 10 and 8% respectively. Nor score lesion of (0) was reported in flock No. 1, while the score was higher ten times in flock No.2.

Table 1: Number of necropsed birds, their percentages and intensity of pathological changes seen in livers of two broiler flocks.

Broiler flocks

%

Intensity

No. of necropsed birds

%

Intensity

2/40

5

+

2/40

5

+

9/40

22.5

++

10/40

5

+

14/40

35

+++

14/40

35

+++

15/40

37.5

++++

14/40

35

++++

1/40

2.5

+

4/40

10

+

11/40

27.5

++

10/40

25

++

13/40

35

+++

12/40

30

+++

15/40

37.5

++++

14/40

35

++++

Sub capsular hemorrhage

Liver lesions

No. of necropsed birds

Flock No. 2

Discoloration (Yellow ocher)

Flock No. 1

Figure 1: Necropsy liver and caecal lesions of broiler 35 days of age, showing creasy, yellow-ocher discolored liver, with ballooned two caeci, filled with free blood.

Parasitological findings of both flocks are summarized in Table 3. The MLS in both flocks was 3.26, with fecal oocyst excretion per gram X1000 = 636. The morphological features of examined caecal oocysts were identical for Eimeria tenella.

Aflotoxin assay of feed commodities taken from both broiler flocks are presented in Table 4. Of these commodities corn samples were heavily contaminated with AF. The range of contamination was between 831-3485 ppb, with average concentration of 1915ppb.Soybean samples were also reported to be contaminated with AF but at a lower rate than that of corn. Similar rate of contamination was noted, with range occurred between 220-238ppb, and an average of 229 ppb. No AF contamination was detected in both wheat and barley commodities. Mixed feed was consequently contaminated from the contaminated ingredients made from them, corn

Individual and mean caecal lesion scores (Mean lesion score, MLS) (MLS = Sum of scores/number of birds) are presented in Table 2. From table it is clear that the total mean score lesions in the first flock was 3.5, i.e., higher than that MLS in the flock N0. 2 (3.05). The distributional percentages of MLS in both broiler flocks are illustrated in Figure 2. From this table it is clear that score 4 represents 73% in flock No.1, compared with 52% in flock N0.2.

Score 3 in flock No. 2 was about double that in flock

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Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (11-16)

Table4: Aflatoxin contamination of feed commodities (ppb) in of both broiler flocks.

and soybean. They were contaminated in a rate of 720-1006 ppb, with an average of 860 ppb.

Table 2: Individual and mean total caecal score lesion of broiler chicks in both flocks.

Caecal score lesion 0 1 2 3 4 Mean lesion score(MLS)

Feed commodities Sample No.

Broiler flocks No. of birds in flock No. 1 4 1 6 29

No. of birds in flock No. 2 4 3 1 10 22

140/40 = 3.5

122/40=3.05

Corn

Soybean

Wheat

Barley

Mixed feed

Concentrations of AF ppb 1 3485 2 3485 3 931 4 831 5 866 Mean 1915 ND= not detected

238 233 228 224 220 229

ND ND ND ND ND ND

ND ND ND ND ND ND

1006 931 831 753 720 860

Discussion

0

1 Flock 2

2

3

4

1

2

3

Caecal score lesions

Aflatoxin was reported to increase susceptibility to coccidiosis under experimentally prior exposure poultry to Aflatoxin (25-30,35). To our knowledge, no report is reported here in Mosul province referring to a natural concurrent secondary caecal coccidiosis due to contamination of broiler feed commodities with Aflatoxin.

In this study, although anticoccidial agent was not included in the diet of broiler flocks as prophylactic measure, but the presence of high natural Aflatoxin contamination levels (860 ppb) of the mixed feed used in the feeding of these flocks, could largely impose a great stress on the birds immune system against coccidial infection.. This relation is supported by (22), who stated that 380 ppb AF or more is responsible for a significant reduction in cell mediated immunity (CMI). This effect on CMI include (a) macrophages, (b) natural killer (NK) cell lymphocytes and (c) two types of T-derived lymphocyte population (CD4+and CD8+) (35,36). Aflatoxin causes a substantial diminution in phagocytic activity by macrophages (37). Macrophages, CD4+ and NK cell lymphocytes dominate the CMI reaction upon primary contact/infection of coccidiosis, while CD8+ cells multiply particularly upon second contact (reinfection) of coccidiosis (38). CD4+ and CD8+ cells are cytotoxic and cytolytic, this means they can kill and lyses parasitic stages during coccidiosis cycle (39). However, it was reported that specific serum antibodies might also play a part in protecting chickens against coccidiosis (40) Antibodies, especially local immunoglobulin A (IgA) in the intestinal lumen can bind and damage sporozoites, causing loss in the ability of extra cellular differentiation and thereby preventing parasite invasion and intracellular development (41). So, antibodies, although play a minor role as a protective component against coccidiosis compared with the role of cell mediated immunity, but it is still negatively

4 Flock 1

Figure 2: Percentages of caecal score lesions in both broiler flocks.

Table 3: Summary of the parasitological examination of necropsed birds (Average findings of broilers in both broiler flocks).

Parameters Mortality Fecal oocyst excretion per gram X1000 Oocyt size width X length (Âľm) Mophological characteristic of eggs Mean lesion score for 80 dead birds Schizonts maximum in Âľm

Results 26% 636 26 X 23 Broadly ovoid 262/80 = 3.27 55.0

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Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (11-16)

affected by Aflatoxin, since their production was reported to be decreased by exposing birds to high toxin levels from 300 to 6000 ppb (42). The other possible roles of AF in predisposing broilers here to coccidial infection is the toxin reduction effect of vitamin A level in chicken liver and serum (43). Here, although vitamin A level was not measured in liver or serum, but it is highly possible that vitamin A deficiency is likely to occur due to feeding highly AF contaminated diet. Since, it is well known that disease resistance is a function of cell differentiation, and one of the primary function of vitamin A is to maintain proper epithelial tissue differentiation and prevent epithelial keratinization which occurs in vitamin A deficiency and it is important for enhancing both cellular and humoral immunity and enhance phagocytosis activity, and for maintaining cellularity of lymphoid organs (44,45). The possible reduction of vitamin A may be related to the high mortality rate reported here, since vitamin A has been reported to reduce mortality in chicks infected with coccidial oocyst from E- tenella and E- acervulina (46).

The high coccidial pathogenicity noticed in our study, expressed by high lesion score of 3.27, could be related, to high AF diet contamination (15), and the high fecal oocyst excretion per gram (636000) oocyst/gram could also be attributed to this contamination. This effect was supported by (2,28), who confirmed that Aflatoxicosis was responsible for increased oocyst production and reproduction potential during coccidiosis. It is evident from the parasitic parameters examined in this study, that lesion score was found to be directly proportional to the mortality and oocyst count per gram of faeces/ dropping. Any way, it should be stressed that other mycotoxins like ochratoxin, T2 toxin, and fumonisins may be present or contaminate feed commodities or mixed feed samples in our study, but the estimation was tried only with the most effective immunosuppressant mycotoxins, Aflatoxin. Moreover, no attempt was carried out to isolate immunosuppressive viruses like reo virus and Merk disease virus to elucidate their possible role if they were present in exacerbating caecal coccidiosis beside aflatoxin on broiler chickens.

From all above, and under field conditions, it seems that mycotoxins and here AF are highly stressful, when fed for extended period of time to birds on their immune system, which may be weakened to the point where it cannot control the organism and set the stage for the development of coccidiosis. So to prevent Aflatoxicosis and coccidiosis in poultry, sorbents like sodium bentonite and hydrated sodium calcium aluminosilicates should be added to their diet in order to decrease Aflatoxin severity (47), and by using coccidiostats/coccidiocidal drugs, or by Appling an alternative system of vaccination against coccidiosis..

Acknowledgement The author thanks the College of Veterinary Medicine, Mosul, Iraq, for providing all supports for this study.

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4. AL-Sadi HI, Shareef AM, AL-Attar MY. Outbreaks of aflatoxicosis in broilers. Iraqi J Vet Sci. 2002;13:93-106.

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7. Henry SH., Bosch FX, Bowers JW. Aflatoxin, hepatitis and worldwide liver cancer risks. Adv Exp Med Biol. 2002;504:229–233.

8. Neldon-Ortiz DL. Direct and microsomal activated aflatoxin B1 exposure and its effects on turkey peritoneal macrophage functions in vitro. Toxicol Appl Pharmacol. 1991;109:432-42.

9. Moon EY. Inhibition of various functions in murine peritoneal macrophages by aflatoxin B1 exposure in vivo. Int J Immunopharmacol. 1999;21:47-58.

10. Cusumano V, Rossano F, Merendino RA. Immunobiological activities of mould products:functional impairment of human monocytes exposed to aflatoxin B sub(1). Res Microbiol. 1996;147:385-91.

11. Liu BH, Yu FY, Chan MH, Yang YL. The effects of mycotoxins, fumonisin B1 and aflatoxin B1, on primary swine alveolar macrophages. Toxicol Appl Pharmacol. 2002;180,197–204.

12. Boonchuvit B, Hamilton PB, Burmeister HR. Interaction of T-2 toxin with salmonella infection of chickens. Poult Sci. 1975;45:1693-1969.

13. Hamilton PB. Anatural and extremely sever Aflatoxicosis in laying hens. Poult Sci. 1971;50:1880-1882.

14. Richard JL, Pier AC, Cysewsk SJ, Granan CK. Effect of AF and aspergillosis on turkey poults. Avian Dis. 1973;17:111-121.

15. AL-Sbawi DMT. Ameliorative effect of mycofix plus.3.0 in reducing intensity of Emeria tenella infection during Aflatoxicosis in broiler chicks (masters thesis) Mosul University; Iraq; 2005.71p.

16. Williams RB. Epidemiological aspects of the use of live anticoccidial vaccines for chickens. Int J Parasitol. 1998;28:1089-1098.

17. Levine ND. Taxonomy and life cycles of coccidian, In: (edited by Long, P. L.) The Biology of the Coccidia, Univeristy Park Press, Baltimore;1982. p.1-33.

18. Witlock DR, Lushbaugh WB, Danforth HD, Ruff M D. Scanning electron microscopy of the cecal mucosa in Eimeria-tenella-infected and uninfected chickens. Avian Dis. 1975;19:293-304..

19. Lillehoj HS. Role of T lymphocytes and cytokines in coccidiosis. Int J Parasitol. 1998;28:1071-1081.

20. Lillehoj HS, Choi KD, Jenkins MC, Vakharia VN, Song KD, Han JY, Lillehoj EP. A recombinant Eimeria protein inducing interferongamma production:comparison of different gene expression systems and immunization strategies for vaccination against coccidiosis. Avian Dis. 2000;44:379-389.

21. Lillehoj HS, Min W, Dalloul RA. Recent progress on the cytokine regulation of intestinal immune responses to Eimeria. Poult Sci. 2004;83:611-623.

22. Williams RB. Intercurrent coccidiosis and necrotic enteritis of chickens: rational, integrated disease management by maintenance of gut integrity. Avian Pathology. 2007;34:159-180.

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23. Williams RB. Efficacy and Epidemiological Aspects of paracox, a new coccidiosis vaccine for chickens. Harefield. UK. Pitman-Morre Europe;1992. 72 p.

24. Ruff MD. Reasons for inadequate nutrient utilization during avian coccidiosis, a review. In McDoughald LR, Joyner LP, Long PL.(Eds).

Research in avian coccidiosis. Athens, University of Georgia;1986:p.

169-185.

25. Wyatt RD, Ruff MD, Page RK. Interaction of aflatoxin with Eimeria tenella infection and monensin in young broiler chickens. Avian Dis. 1975;19 :730-740.

26. Edds GT, Simson CF. Cecal coccidiosis in poultry as affected by prior exposure to aflatoxin B1. Am J Vet Res. 1976;37:65-68.

27. Rao JR, Sharma NN, Iyer PK, Sharma AK. Interaction between Eimeria uzura infection and aflatoxicosis in Japanese quail (Coturnix coturnix japonica).Vet Parasitol. 1990;35:259-267.

28. Awadalla SF. Influence of dietary aflatoxin on the severity of coccidial infection in quails. Egypt Soc Parasitol. 1999;28:437-447.

29. Ruff MD, Fagan JM, Dick JW. Pathogenicity of coccidian in Japanese quail(Coturnix coturnix japonica). Poult Sci. 1984;63:55-60.

30. Ruff MD. Influence of dietary aflatoxin on the severity of Eimeria acervulina infection in broiler chickens. Avian Dis. 1978;22:471-480.

31. AL-Taee AFM. Incidence of broiler cecal coccidiosis in Ninevah governorate 1999-2004. Iraqi J Vet Sci. 2007;21:45-65.

32. Johnson J, Ried WM. Anticoccidial drugs:Lesion scoring techniques in battery and floor-pen experiments with chickens. Exp Parasitol.

1970;28:30-36.

33. Foreyt WJ. Veterinary parasitology. Reference manual.5th ed. Iowa State University Press. USA.2001.307p.

34. Smith JE, Moss MO. control of mycotoxins. Ltd, London, John Wiley and Sons;1985. 142 P. 35. Witlock DR, Wyatt RD, Anderson WI. Relationship between Eimeria adenoids infection and Aflatoxicosis in turkey poults. Poult Sci. 1982;61:1293-1297

36. Ghosh RC, Chauhan HVS, Jha GJ. Suppression of cell-mediated immunity by purified aflatoxin B1 in broiler chicks. Vet immunol immunopathol. 1991;28:165-172.

37. Lillehoj HS, Min W, Dalloul RA. Recent progress on the cytokine regulation of intestinal immune responses to Eimeria. Poult Sci. 2004;83:611-623.

38. Qurshi MA, Brake J, Hamilton PB, Hagler JR WM, Nesheim S. Dietary exposure of broiler breeders to aflatoxin results in immune dysfunction in progeny chicks. Poult Sci 1998;77:812-819 39. Breed DGJ, Dorrestein, Vermeulen AN. Immunity to Eimeria tenella in chickens: Phenotypical and functional changes in peripheral blood T cells subsets. Avian Dis. 1996;40:37-48.

40. Davison TF, Morris TR, Payne LN. Poultry immunology. Poultry science symposium series vol.24. Journals oxford ltd;1996. 300 p.

41. Bakshi CS, Sikdar A, Johri TS, Malik M. Effect of graded dietary levels of AF on cell mediated immune response in broilers. Indian J Comp Microbiol,Immunol and Inf Dis. 1998;19:40-42 42. Yun CH, lillehoj HS, Lillehoj EP. Intestinal immune responses to coccidiosis. Dev Com Immunol, 2000;24:303-424.

43. Pimpukdee K, Kubena LF, Bailey CA, Huebner HJ, Afriyie-Gyawu E, Phillips TD. Aflatoxin-induced toxicity and depletion of hepatic vitamin a in young broiler chicks:protection of chicks in the presence of low levels of novasil plus in the diet. Poult sci. 2004;83:737â&#x20AC;&#x201C;744.

44. Davis CY, Sell JL. Effect of all-trans retinol and retinoic acid nutrition on the immune system in chicks. J Nutr. 1983;113:1914-1919.

45. Latshaw DJ. Nutrition-mechanisms of immunosuppression. Vet Immunol Immunopathol. 1991;30:111-120 46. Erasmus J, Scott ML, Levine PP. A relationship between coccidiosis and vitamin A nutrition of chickens. Poult Sci. 1960;39:565-572.

47. Shareef AM. The effect of two commercial bentonites in improving broiler performance during Aflatoxicosis. Iraqi J Vet Sci. 2004;18: 101-109.

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Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (17-25)

Molds and mycotoxins in poultry feeds from farms of potential mycotoxicosis A. M. Shareef Department of Veterinary Public Health, College of Veterinary Medicine, University of Mosul, Mosul, Iraq (Received June 19, 2008; Accepted July 13, 2009) Abstract Forty five finished poultry feed samples, collected from different broilers, broiler breeders and layers farms were divided into two parts, for mycological and mycotoxins examination. In counting of molds, dilute plate technique was used, whereas feed parts were used for mycotoxin estimation, they were subjected to four standard kits of Aflatoxin, Ochratoxin, T-2 toxin and Fumonisins. Mold counts were around 105 cfu.g-1 sample. Fourteen mold genera were recovered. From the systematic point of view, 2 genera belonged to Zygomycetes (i.e. Mucor, Rhizopus,), 1 genus belong to Ascomycetes (i.e. Eurotium); the majority, within so-called mitotic fungi (formerly Deuteromycetes), encompassed 11 genera (i.e. Acremonium, Alternaria, Aspergillus, Fusarium, Paecilomyces, Penicillium, Scopulariopsis,, Trichothecium, Ulocladium and Aerobasidium). The most frequent fungi were those from the genus Aspergillus. The concentrations of the four analyzed mycotoxins in the poultry finished feeds, and the percentages of the recovered mycotoxins, revealed that aflatoxins was recovered in 91.1% of the examined samples, with a mean value of 179.1Âľg/kg. The same percentage was found with Ochratoxins, but with lower mean concentration of 159.4Âľg/kg. In the third order were Fumonisins mycotoxins were in the third order, and they were recovered in 51.1% of the tested samples with a mean value of 127Âľg/kg. In the fourth order was T-2 toxin, with a percentage of 2.2% and a value of 50.0Âľg/kg.

Key words: Molds, Mycotoxins, Mycotoxicosis, Poultry feed.

Available online at http://www.vetmedmosul.org/ijvs

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Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (17-25)

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GAL governorate/ Iraq that It includes: enumeration and identification of moulds genera naturally contaminating different kinds of poultry feeds and detection and quantification of the 4 major mycotoxins in the feed samples, aflatoxin, ochratoxin A, fumonisins and T-2 toxin.

Introduction Mold occurrence and growth on poultry feeds is one of the major threats to poultry economic and health. Besides their negative impacts on nutritional and organoleptic properties, moulds can also synthesize different mycotoxins. More than 100.000 fungal species are considered as natural contaminants of agricultural and food products. However, due to genetical and ecological factors, relatively few can actually generate mycotoxins (1).

According to Leibetseder (2), 30 to 40 % of existing moulds can elaborate toxic substances under favorable conditions. The majority of the toxic species belong to the genera Aspergillus, Penicillium, Fusarium and Alternaria (3). The effects of mycotoxins on higher animals include hepatotoxicity, nephrotoxicity, immunotoxicity, oncogenesis and genotoxicity (4-6). Despite the great attention that has been paid to the study of toxigenic moulds and their mycotoxins in various foods and feeds, little is known about fungal and mycotoxin contamination of poultry feed in Mosul governorate yet. During the last two or three decades, the production of mixed poultry feeds were significantly increased in parallel with the evolution of poultry industry in the country. Furthermore, it is well established that contamination of poultry feeds with mycotoxins may induce sanitary disturbances and mortality among the birds and secondary contamination of the human consumer via eggs, poultry meat and giblets (7,8). Variety of complex and divers clinical signs of potential mycotoxicosis were always observed in different broiler, broiler breeder and layer farms with potential mycotoxicosis. Affected flocks showed one or more of the following symptoms; decreased weight gain; anorexia; reduced feed conversion efficiency; decreased egg production; poor egg shell quality ;increased egg blood spots; spiking mortalities; immunosuppression and failure of vaccination programs; increased susceptibility to diseases especially E-coli infection; reduced fertility and hatchability; visceral hemorrhages; leg weakness and high percentages of leg deformities; pale bird syndrome; fatty liver with pale, muddy to yellowish discoloration; increased bruising; enlarged pale kidneys; wet litter; urate deposition in the body cavities; increased incidence of viral diseases like Newcastle disease, infectious bursal disease and inclusion body hepatitis; oral lesions; tibial dyschondroplasia; gizzard erosions; paralysis; extension of leg and neck.

The purpose in the present work was to initiate a study on the toxigenic mycoflora of poultry feed in Ninevah

Materials and methods Samples The samples of finished poultry feed were delivered to the College of Veterinary Medicine, University of Mosul from different broilers, broiler breeders and layers farms.

These farms were located in Nineveh governorate and were claimed from potential mycotoxicosis, feed samples were collected during a 2-years-long period from Apr 2005 to Sep 2007. Random 10 to 30 representative samples of I kg were collected from several locations within a batch of feed and combined thoroughly to provide a composite sample of 1 Kg for submission. They were then divided to two parts, one for mycological examination, and the other for mycotoxins detection. Feed samples intended for mycological examination were usually analyzed immediately upon arrival or, if necessary, they were stored for 2-3 days in paper bags at room temperature (22-25ยบC).

The other parts of feed samples intended for mycotoxins analysis were stored at -20Co. Isolation of poultry feed fungi Dilute plate technique was used for isolation of fungi from the samples (9). General molds count was carried out by weighing 20 g of the poultry mixed feed samples and their mixing with 180 ml of saline solution (0.85% sodium chloride) with 0.05% Tween 80 on a horizontal shaker for ca. 30 minutes. Then, 0.1 ml of appropriate dilutions made up to 10-5 was applied on Dichloran Rose Bengal Chloramphenicol agar (DRBC (10, 11). Plates were incubated at 25oC for 5-7 days. The mold genera were identified according to (11-15).

Mycotoxins analyses Feed parts intended for mycotoxin estimation were subjected to four standard kits for aflatoxin, ochratoxin, T-2 toxin and fumonisins using Neogen mycotoxin extraction kits (Neogen corporation) as follows: Samples preparation and extraction: Twenty five gramsamples were collected for analysis. These samples were finely ground,so that at least 75% of them pass through a 20 mesh. Five gram samples were blended with 25 ml of 70%

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v/v methanol/ water solution for 3 minutes. Extracts were filtered through a Whattman No.1 filter paper.

Test procedure: All Neogen extraction reagents were allowed to warm at room temperature (18-30 ºC) before use. Red marked mixing wells were prepared, one for each sample plus four red wells for controls. All red- marked wells prepared were placed in the well holder. An equal number of antibody coated (AB) white wells to those redmarked wells were also prepared. Hundred µl of conjugate were transferred to each red-marked mixing well. To those red wells containing the conjugate another (with new pipette tips) 100 µl of controls and samples were added by using a 12-cannel pipettor liquid in wells were mixed and pipetting it up and down for 3 minutes. After mixing 100 µl of the (conjugate + samples, or conjugate + controls) were transferred to AB – coated wells. These wells were moved back and froth for well mixing the contents in each well for 10-20 seconds without splashing reagents from the wells.

Antibody-coated wells were then incubated at room temperature (18-30 ºC). The contents in AB- coated wells were shacked out, by filling the wells with deionized water and dumping them out. This step was repeated 5 times.

Turning the wells upside down and tamping them out on a paper was carried out until the remaining water has been removed. Substrate was then added to AB- coated wells, by using the 12-channel pipettor through pipetting 100 µl of substrate to these wells. Mixing was done by sliding the well holder back and froth for 10-20 seconds, followed by incubation for 3 minutes. Stop solution was poured to these wells (100 µl) to each, mixing was done by sliding well holder back and froth on a flat surface. Within 20 minutes after the addition of stop solution. Results were read, using a micro well reader (ELx00) with a 650 nm filter. More blue color means less toxins. Results of the yield optical densities of the controls and samples were obtained by using computerized Neogen Verotex Softwere Program version 2.9.16 (Neogen Corporation).

frequent fungi were those from the genus Aspergillus, recovered from 40 samples (88%) with a range of 0.1 X 10 5 - 5.3 X 106 and a mean value of 2.6 X 106. In the second order were the molds from the genus Penicillium and Mucor, both recovered from 28 samples (64%) with a range of 0.2 X 105 - 4.4 X 106 and3 X 104 - 2.6 X 105 and a mean of 2.2 X 106 and 1.4 X 105 respectively. To the third and fourth orders were the molds from the genera Rhizopus, and Scopulariopsis, recovered from 23 and 22 samples (50% and 48%) respectively with a range of 1.5 X 104- 2.1 X 105 and 0.2 X 104- 1.8 X 105, and a mean of 1.1 X 105 and 9.1X 105 respectively. The following recovered genera were the molds of Alternaria and Eurotium, from 20 and 19 samples with percentages of 45% and 42% and a range of 8 X 10 3 1.8 X 1053 and X 10 3 - 1.7 X 105, with a mean of 9.4 X 104 and 8.5 X 104 respectively. In the descending pattern occurred the molds of the genera Cladosporium and Fusarium, recovered from 17 and 14 samples (37% and 31%), representing a range between0.2 X 10 3- 9.8 X 104 and 0.4 X 10 3 - 9.2 X 104, and a mean of 4.9X 104 and 4.6 X 104 respectively. From less than 10 samples recovered the genera of the mitotic molds from Acremonium,, Paecilomyces, Ulocladium, Aureobasidium, and Trichothecium, with 11%,7%,7%,2% and 2% respectively, with an average of 4300,110,70,50 and 25 CFU g1respectively.

Table 2 illustrate the frequency of recovered mold genera out of 45 finished poultry feeds tested through the study period, of these 41 samples were positives for aflatoxins and ochratoxins (91.1%).To a lower extent were the positive samples of Fumonisins (51.1%). The lowest positive sample was that of T-2 Toxin (2.2%). The mycotoxins concentrations were in general positive proportion to the number of respective molds producing them, being highest in aflatoxins and ochratoxins. These mycotoxins are produced mainly by aspergillus and penicillium mold genera, the most prominanat mold enumerated in this study. In a descenging manner were also the average levels of aflatoxins, ochratoxin A, fumonisins and T-2 toxin. The highest levels of these mycotoxins were as follows; aflatoxins: < 475 µg/kg ; ochratoxin A: < 460 µg/kg ; fumonisins: < 350 µg/kg and T-2 toxin: 50 µg/kg.

Table 3 shows the distribution concentration of different mycotoxins in the tested finished poultry feeds.

Only aflatoxins and ochratoxin A were ranged from 0-500 µg/kg. The highest number of samples contaminated with aflatoxin were within levels of 200-300 µg/kg. Although ochratoxin level was also ranged from 0-500 µg/kg, but the highest levels were occurred in concentrations of 50 and 250 µg/kg. Fumonisins highest levels were occurred in concentrations between 50-150 µg/kg, and the highest levels did not exceeds 350 µg/kg. The only one sample contaminated with T-2 toxin had level of 50 µg/kg.

Results The total number and percentages of enumerated mold genera in Nineveh finished poultry feed are presented in Table 1, and the ability to produce appropriate mycotoxins is shown in Table 2. Total fungal counts were ranged from 0.1 × 101 to 6.5 X 106 cfu.g-1 of feed sample, with an average 7.2 X 105 cfu.g-1 sample (Table 1. Fourteen mold genera were recovered during this study.

From the systematic point of view, 2 genera belong to Zygomycetes (i.e. Mucor, Rhizopus,), 1 genus belong to Ascomycetes (i.e. Eurotium); the majority, within so-called mitotic fungi (formerly Deuteromycetes), encompassed 11 genera (i.e. Acremonium, Alternaria, Aspergillus, Fusarium,, Paecilomyces, Penicillium, Scopulariopsis,, Trichothecium, Ulocladium and Aerobasidium). The most

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Table 1: Frequency and average count of recovered mold genera from 45 Nineveh poultry finished feed samples.

Molds Aspergillus spp.

Penicillium spp.

Mucor spp.

Rhizopus spp.

Scopulariopsis spp.

Alternaria spp.

Eurotium spp.

Cladosporium spp.

Fusarium spp.

Acremonium spp.

Paecilomyces spp.

Ulocladium spp.

Trichothecium spp.

Aureobasidium spp.

Frequency of Positive samples 40 28 29 23 22 20 19 17 14 5 3 3 1 1

Percentage of mold genera frequencies 88.8 62.2 62.2 51.1 48.8 44.4 42.2 37.7 33.3 11.1 6.6 6.6 2.2 2.2

Range 0.1 X 10 5- 5.3 X 106 0.2 X 105 - 4.4 X 106 3 X 104 - 2.6 X 105 1.5 X 104- 2.1 X 105 0.2 X 104- 1.8 X 105 8 X 10 3 - 1.8 X 105 3 X 10 3 - 1.7 X 105 0.2 X 10 3- 9.8 X 104 0.4 X 10 3 - 9.2 X 104 0.6 X 10 2 - 8.7 X 103 1 X 10 1 - 2.1 X 102 2 X 10 1 - 1.2 X 102 0.2 X 10 1 - 0.8 X 101 0.2 X 10 1 - 0.8 X 101

Average counts cfu g-1 2.6 X 106 2.2 X 106 1.4 X 105 1.1 X 105 9.1X 105 9.4 X 104 8.5 X 104 4.9X 104 4.6 X 104 4.3 X 103 1.1 X 102 7 X 101 5 X 101 2.5 X 101

Table 2: Number of tested, positive, percentage and levels of detected mycotoxins in PMFS tested.

Parameters No. of samples tested No. of positive samples Percentage positive (%) Average level (Âľg/kg) Highest level (Âľg/kg)

Mycotoxins Ochratoxin A Fumonisins 45 45 41 23 91.1 51.1 159.4 127 460 350

Aflatoxins 45 41 91.1 179.1 475

Figure 4 shows the different AF concentrations (ppb) in positive AF-PFF samples. It is evident that 24% of the samples had AF concentration between 201-250 ppb, followed by 16.6% between 151-200ppb and 11.1% between 351-400 ppb. The remaining concentrations were recovered in less than 10% of the samples.

Figure 5 shows the different Ochratoxin concentrations (ppb) in positive Ochratoxin -PFF samples. From figure it is clear that 20% of the samples had Ochratoxin up to 50 ppb, followed by 17.7% between 201-250 ppb, 13.3% between 51-199 and 351-400, 11.1% between 101-150, while the remaining concentration were occurred in percentages less than 10%.

Figure 6 shows the different fumonisins concentrations (ppb) in positive fumonisins -PFF samples. It is evident that 48.8% were negative samples. The positive samples showed that 22% of them had fumonisins concentrations up to 50 ppb, and 13.3% between 101-150 ppb, while the concentrations up to 350 ppb had percentages of less than 10%. No more than 350 ppb of fumonisins mycotoxin was detected.

Figure 7 shows that 97.75 of the tested feed samples were negative for T-2 toxin, and only one sample with 50 ppb was detected.

T-2 Toxin 45 1 2.2 50 50

Discussion Both field and storage fungi were recovered in this study through the examination of poultry finished feed samples collected from different poultry farms with potential mycotoxicosis. These molds include members of the genera Aspergillus, Penicillum, Fusarium, Alternaria and Cladosporium which could contaminate many agricultural commodities used in the formulation of poultry finished feed samples like corn, wheat, soybean, barley and others commodities. These molds are of great importance because of potential mycotoxin production (16). The total fungal loads in the analyzed finished feed samples in our study were around 105 cfug-1 which is higher than that reported in Slovakia of 103 cfu.g-1 (17), and 104 cfu.g-1 (18). Similar results were found to those reported in Turky (19), Spain (20) and from Argentina (21,22). The predominance of mold genera were to large extent resembles those found in Saudi Arabia (23), which could be due to some resemblance in the ecological and climatologically conditions. The main contaminating molds appeared to be from the genus Aspergillus with a percentage recovery of 91.11%.

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Table 3: Mycotoxins recovered from Nineveh poultry finished feed.

This finding in consistent with (22) and (24) and (25) who found that the most dominant species isolated of poultry feed samples belonged to the genus Aspergillus, but not in consistent with (17), (22) and (25), who found that the most frequent fungi were those from the genus Penicillium.

Although molds of Aspergillus species are more often soil fungi or saprophytes but several are important because they produce mycotoxins. Penecilli in our results were in the second order in counting and percentages among recovered molds, as they are one of the main contaminants of stored cereals as well as feeds with worldwide occurrence (26-28) being producers of toxicologically significant mycotoxins (29-31). In order, our results were in consistent with (17) that the third mostly encountered fungi were representatives of the genus Mucor, who found this genus with frequency of 44%, followed by the genus Rhizopus. Our results also revealed that molds from the genus Eurotium were recovered in 19%, and Fusarium in 14% of the samples.

These results were less than that reported by (17), who found that Eurotium and Fusarium genera were widespread through the samples they examined and were occurred with the same frequency of 42%. In this study, the mean of aflatoxins were recovered at a rate of 91.1%, with an average of 179.1µg/kg of finished poultry feed samples, confirming our previous results of aflatoxin contamination to poultry feed samples (32). Comparing with other Asian countries, our results were higher than that reported in north Asia (China, Taiwan and Korea) of 3%, and higher than south East Asia (Malaysia, Philippines, Thailand and Vietnam) of 36%, and higher than south Asia (India, Pakistan and Bangladesh) of 53% (33). The second mycotoxin recovered was Ochratoxin (91.1%), in an average of 159.4 µg/kg, confirming our previous results of ochratoxin contamination of poultry finished feeds (34).

Our results were also more than those reported in north Asia (China, Taiwan and Korea) of 15%, and higher than south East Asia (Malaysia, Philippines, Thailand and Vietnam) of 9%, and higher than south Asia (India, Pakistan and Bangladesh) of 50% (33). Again our high results here were due to sampling feeds from mycotoxicosis suffering poultry farms. Ochratoxin has been implicated in significant field outbreaks of mycotoxicosis in poultry (35).

Fumonisins mycotoxins were the third type of mycotoxins recovered in this study, in a percentage of 53.3% and an average of 127µg/kg of finished poultry feed samples.

These mycotoxins recovered here in Mosul governorate in corn samples intended for use in finished poultry feeds (36). Our results agreed with the percentages reported in north Asia (China, Taiwan and Korea) of 52%, and with those of south East Asia (Malaysia, Philippines, Thailand and Vietnam) of 52%, but higher than hose reported in south Asia (India, Pakistan and Bangladesh) of 32% (33).

The fourth mycotoxin recovered in this study was T-2 toxin in a percentage of 2% at a rate of 50 µg/kg. This toxin was

T-2 toxin ppb PFF 1* 57 30 ND ND** PFF 2 189 290 100 ND PFF 3 254 230 ND ND PFF 4 370 61 ND ND PFF 5 69 291 ND ND PFF 6 165 242 200 ND PFF 7 ND. 159 250 ND PF F8 250 ND. ND ND PF F9 350 42 ND ND PF F10 452 460 200 ND PFF 11 54 91 ND ND PFF 12 152 420 ND ND PF F13 250 223 150 ND PFF 14 340 133 150 ND PF F15 ND. 173 30 ND PF F16 458 ND. ND ND PFF 17 146 228 ND ND PFF 18 ND 191 150 ND PF F19 240 ND. ND ND PF F20 36 133 200 ND PFF 12 362 23 350 ND PFF 22 475 214 ND ND PFF 23 76 38 ND ND PF F24 158 28 150 ND PF F25 370 212 30 ND PF F26 356 273 ND ND PF F27 235 ND. 20 ND PFF 28 230 19 50 ND PF F29 235 12 130 ND PFF 30 453 73 20 ND PF F31 ND. 260 ND ND PFF 32 293 142 20 ND PF F33 250 212 ND ND PF F34 323 57 ND ND PF F35 230 72 ND ND PF F36 196 12 250 ND PF F37 226 68 ND ND PF F38 180 50 20 ND PFF 39 400 252 250 ND PFF 40 196 222 10 ND PF F41 188 142 ND ND PFF 42 250 123 20 ND PF F43 219 183 ND ND PF F44 90 280 150 ND PF F45 22 320 150 50 *PFF=Poultry finished feed from farm 1 ND**=Not detect.

Sample Aflatoxins Ochratoxins No. ppb ppb

Fumonisins ppm

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Figure 4:Distribution of different AF concentrations(ppb) in positive AF- PFF samples 30 25

24.4%

15

11.1%

%

20

16.6%

10 5 0 11

10

9

8

7

6

5

4

3

2

1

Concentrations

1=ND 2=0-50ppb 3=51-100ppb 4=101-150ppb 5=151-200ppb 6=201-250ppb 7=351-300ppb 8=301-350ppb 9=351-400ppb 10=401-450 ppb 11=451-500ppb

Figure5: Distribution of different Ochratoxin concentrations (ppb) in positive Ochratoxin-PFF samples 25 20%

13.3%

20

13.3%

15

11.1%

10 5 0 11

10

9

8

7

6

5

4

3

2

1

Concentrations

1=ND 2=0-50ppb 3=51-100ppb 4=101-150ppb 5=151=200ppb 6=201-250ppb 7=351-300ppb 8=301-350ppb 9=351-400ppb 10=401-450 ppb 11=451-500ppb

22

%

17.7%

Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (17-25)

Figure 6:Distribution of different Fumonisins (ppb) concentrations in positive Fumonisins-PFF in PFF samples 60 48.8%

22.2%

30

%

50 40 20 10

13.3%

0 11

10

9

8

7

6

5

4

3

2

1

Concentrations

1=ND 2=0-50ppb 3=51-100ppb 4=101-150ppb 5=151=200ppb 6=201-250ppb 7=351-300ppb 8=301-350ppb 9=351-400ppb 10=401-450 ppb 11=451-500ppb

Figure 7:Distribution of different T-2 toxin (ppb) in positive T-2 toxin - PFF samples 120 100

97.7%

60 40 20

2.2%

0 11

10

9

8

7

6

5

4

3

2

1

Concentrations

1=ND 2=0-50ppb 3=51-100ppb 4=101-150ppb 5=151=200ppb 6=201-250ppb 7=351-300ppb 8=301-350ppb 9=351-400ppb 10=401-450 ppb 11=451-500ppb

23

%

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6. Kurata A, Ueno Y. Toxigenic fungi: Their toxins and health hazard.

Odansha-Elseviers Tokyo-Amsterdam-Oxford-NewYork; 1984. 247 p.

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8. Naoom RAF. Estimation of aflatoxin residues for some ruminant and poultry (Local and imported) livers in Mosul. (masters thesis) Mosul; 2007. 94 p.

9. Edmund M., Roger L. and Yoshito M.: Microbiology of processed spices. J Milk Food Technol. 1975;38:683-687.

10. Pitt JI, Hocking AD. Fungi and food spoilage. Sydney: Academic press; 1985. 185p.

11. Pitt JI. Penicillium viridicatum, Penicillium verrucosum and production of ochratoxin A. Appl Environm Microbiol. 1987;53:266269.

12. Pitt JI. The genus Penicillium and its teleomorphic states Eupenicillium and Talaromyces. Academic Press. London; 1979. 206p.

13. Pitt JI. Laboratory guide to common Penicillium species. Academic Press London, 1988. 87p.

14. Raper K, Fennel DI. The genus Aspergillus. The Williams and Wilkins co, Baltimore, U.S.A. 1965. 306p.

15. Domsch KH, Gams W. Compendium of soil fungi. Academic Press.

London, New York, Toronto, Sydney, San Francisco; 1980. 566p.

16. David MW, Wellington M, Zeljko J. Biology and ecology of mycotoxigenic Aspergillus species as related to economic and health concerns, In: Mycotoxins and food safety, DeVries W, Truksess W, Jackson LS. Advances in experimental medicine and biology. Kluwer Academic/Plenum Publishers.USA.2002;504:3-17.

17. Labuda R, Tančinová D. Fungi recovered from Slovakian poultry feed mixtures and their toxinogenity. Ann Agric Environ Med. 2006;13: 193–200 18. Magnoli CE, Saenz MA, Chiacchiera SM, Dalcero AM. Natural occurrence of Fusarium species and fumonisin-production by toxigenic strains isolated from poultry feeds in Argentina. 1999;145: 35-41 19. Heperkan D, Alperden I. Mycological survey of chicken feed and some feed ingredients in Turkey. J Food Protect. 1988;51:807-810.

20. Bragulat MR, Abarca ML, Castella G, Cabanes FJ. A mycological survey on mixed poultry feeds and mixed rabbit feeds. J Sci Food Agric. 1995;67:215-220.

21. Dalcero A, Magnoli C, Chiacchiera S, Palacios G, Reynoso M. Mycoflora and incidence of aflatoxin B1, zearlaenone and deoxynivalenol in poultry feeds in Argentina. Mycopathologia. 1997; 137:179-184.

22. Dalcero A, Magnoli C, Luna M, Ancasi G, Reynoso MM, Chiachiera S, Miazzo R, Palacio G. Mycoflora and naturally occurring mycotoxins in poultry feeds in Argentina. Mycopathologia. 1998;141: 37-43.

23. Abdul Wahab R. Hashem. Seed-borne fungi in domestic bird feed in Saudi Arabia. 1996;37:223-226.

24. Pacin AM, González HHL, Etcheverry M, Resnik SL, Vivas L, Espin S. Fungi associated with food and feed commodities from Ecuador.

2003;156:87-92.

25. Glenda RO, Jessika MR, Marcelo EF, Lilia RC, Gloria MD, Kelly MK, Ana MD, Carlos AR. Mycobiota in poultry feeds and natural occurrence of aflatoxins, fumonisins and zearalenone in the Rio de Janeiro State, Brazil. 2006;162:355-362.

26. Filtenborg O, Frisvad JC, Samson RA. Specific association of fungi to foods and influence of physical environmental factors. In: Samson RA, Hoekstra ES, Frisvad JC, Filtenborg O (Eds): Introduction to Foodand Airborne Fungi,. Centraalbureau voor Schimmecultures, Utrecht 2002. 306-320p.

27. Leistner L. Toxinogenic penicillia occurring in feeds and foods. A review. Food Technology in Australia. 1984;36:404-413.

28. Pitt JI. Biology and ecology of toxigenic Penicillium species. In: Trucksess et al. (Eds): Mycotoxins and Food Safety,. Kluwer Academic Plenum Publishers, London 2002. 29-41p.

also recovered by us through the study performed during 2004-2005 in Mosul governorate (37). Our results were close to those reported in north Asia (China, Taiwan and Korea) of 0%, and those found in south East Asia (Malaysia, Philippines, Thailand and Vietnam) of 1%. In general our results were agreed with those reported by Pacin et al., (38) who found that the most recovered mycotoxins from feed associated fungi were ochratoxin A, aflatoxin and fumonisin. It may be stated that Aspergillus (including Eurotium), Penicillium and Fusarium are the typical fungal genera inhabiting poultry feed mixtures. In fact, they are very important contaminants being renowned for their ability to form a huge number of various types of toxic extrolites-mycotoxins (39,40), and that the outcomes of this study clearly show that finished poultry feeds in Nineveh governorate represent a rich source of significant mycotoxin producers, especially those from the Penicillium, Fusarium and Aspergillus genera. It is possible that these co-contaminant of estimated mycotoxins in our study, of two carcinogenic mycotoxins, aflatoxin and fumonisins, and two cancer promoting mycotoxins, ochratoxin and T-2 toxin, could exert great negative effects on farms health and productivity than do each of them singly and of public health concern to the health of consumer through the food chain by ingestion residual levels of these toxins in poultry meat and poultry products (meat and eggs) (41,42). These toxic substances are known to be either carcinogenic (e.g. aflatoxin B1, fumonisin B1, Ochratoxin A), neurotoxic (fumonisin B1), nephrotoxic (ochratoxin A), dermatotoxic (trichothecenes), or immunosuppressive (aflatoxin B1, ochratoxinA and T-2 toxin) (33). the senerginestic effects of these mycotoxins on poultry productivity and health were well documented.(4345).

Acknowledgements The author thanks the College of Veterinary Medicine, University of Mosul for providing all supports for this experiment.

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29. Frisvad JC, Filtenborg O: Terverticillate Penicillia. Chemotaxonomy and Mycotoxin Production. Mycologia. 1989;81:837-861.

30. Frisvad JC, Smedsgaard J, Larsen TO, Samson RA. Mycotoxins, drugs and other extrolites produced by species in Penicillium subgenus Penicillium. Stud Mycol. 2004;49:201-242.

31. Frisvad JC, Thrane U. Mycotoxin production by common filamentous fungi. In: Samson RA, Hoekstra ES, Frisvad JC, Filtenborg O (Eds).

Introduction to Food- and Airborne Fungi,. Centraalbureau voor Schimmecultures, Utrecht 2002. 321-330p.

32. C.A.S.T. Mycotoxins:Economic and health risk.Task force report No. 116. Concil for Agricultural Science and Technology. Ames. IA. USA. 1989. 1-91p.

33. Lee-Jiuan C, Li-Mien T. High occurrence of mycotoxins in Asian feedstuffs. World poultry. 2006;10,4:13-16.

34. Shareef AM. Natural contamination of some broilers feed commodities with ochratoxin, Iraqi J Vet Sci. 2006;20:9-17.

35. Leeson S, Diaz G, Summers JD. Poultry metabolic disorders and mycotoxins. University books. P.O. Box 1326. Canada.1995. 277248p.

36. Shareef AM. Fumonisns mycotoxin contamination level of corn meal used for poultry feeding. Accepted in Raf J Sci. 2008: Under publication 37. Shareef AM. Natural occurrence of T-2 toxin in broiler’s feed commodities determined with Elisa. Iraqi J Vet Sci. 2007;21:167-173.

38. Pacin AM, González HHL, Etcheverry M, Resnik SL, Vivas L, Espin S. Fungi associated with food and feed commodities from Ecuador.

2003;156:87-92.

39. Samson RA. Polyphasic taxonomy of Penicillium subgenus Penicillium. A guide to identification of food and air-borne terverticillate Penicillia and their mycotoxins. Stud Mycol. 2004;49:1173.

40. Frisvad JC, Thrane U. Mycotoxin production by common filamentous fungi. In: Samson RA, Hoekstra ES, Frisvad JC, Filtenborg O (Eds): Introduction to Food- and Airborne Fungi,. Central bureau voor Schimme cultures, Utrecht. 2002. 321-330p.

41. Hiba SA. Estimation of aflatoxin levels for some raw milk types and cheeses (Local & imported) in Mosul city. (Masters thesis) Mosul.

Iraq. 2007.115 p.

42. Whitlow L. Mycotoxins are a bombshell in distiller's grains. World Poultry. 2007;23:13 43. Huff WE, Harvey RB, Kubena LF, Rottinghaus GE. Toxic synergism between aflatoxin and T-2 toxin in broiler chickens. Poult Sci. 1988; 67:1418–1423.

44. Kubena LF, Harvey RB, Buckley SA, Edrington TS, Rottinghaus GE. Individual and combined effects of moniliformin present in Fusarium fujikuroi culture materialand aflatoxin in broiler chicks. Poult Sci. 1997;76:265–270.

45. Harvey RB, Kubena LF, Rottinghaus GE, Turk JR, Harvey HH, Kubena LF. Fujikuroi culture material and deoxynivalenol from naturally contaminated wheat incorporated into diets of broiler chicks.

Poult Sci. 2004;41:957–963.

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Prevalence of intestinal ciliate Buxtonella sulcata in cattle in Mosul T. M. Al-Saffar*, E. G. Suliman**, H. S. Al-Bakri** *Department of Internal and Preventive Medicine, **Department of Microbiology, College of Veterinary Medicine, University of Mosul, Mosul, Iraq (Received June 11, 2008; Accepted July 13, 2009) Abstract The current study was conducted to detect the presence of Buxtonella sulcata (an intestinal ciliate) in faecal samples of cattle suffering from diarrhea in Mosul city. One hundred and twenty faecal samples were examined, and collected from calves (44), beef cattle (34) and dairy cattle (42) these animals were divided into two groups those showed diarrhea (86) and (34) had no symptomatic diarrhea. Direct smear and formalin-ether sedimentation methods were used for detection of this parasite. The total percentage of infection with Buxtonella sulcata was 24.16%. There was no significant differences in the percentage of infection and intensity of infection between calves, beef and dairy cattle where as there were significant differences between diarrheic and non-diarrheic animals.

Keywords: Buxtonella sulcata, Diarrhea, Cattle.

Available online at http://www.vetmedmosul.org/ijvs

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Kinetofragminophora, order: Trichostromatida, Family: Pyenotrichidae, Genus: Buxtonella (1).

Henrisken (7) reported that percentage of infection with Buxtonella sulcata in Danish cattle was (71.8%), Fox (2) recovered Buxtonella sulcata cysts in faeces of adult British cow from nine commercial dairy farms with a percentage of 44.6% and in Kynoggi-don the prevalence of Buxtonella sulcata during 1984 and 1994 were 33.6%, 34.5% respectively (8). A higher percentage 38.0%, 21.6% was reported in a dairy and beef cattle in Costa Rica (9). In

Introduction Buxtonella sulcata is one of the parasitic protozoa (Ciliophora) type which inhabited in the colon of the ruminants and the original role for diarrhea or for classification has not been fully explained (1-5). Buxtonella sulcata is similar to Balantidium coli found in the swine and man, some authors included them into the same genus (6). However, the classification of Buxtonella sulcata is: Kingdom: Protozoa, phylum: Ciliophora, Class:

27

Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (27-30)

Germany a more controversial prevalence was reported which has been range from Zero-73% without any obvious association with clinical symptoms (10). The first recorded of infection with Buxtonella sulcata from cattle was in Al Qadissiyh city (Iraq) and the percentage of infection was 47% (11). The problem of Buxtonellosis in ruminants is not considered of importance yet, therefore the aim of this study was to diagnose this parasite in faeces of cattle in Mosul city and to examine the role of this parasite as etiological agent of diarrhea in calves, dairy and beef cattle.

Results During coproscopical examination of 120 faecal samples of cattle, infection with Buxtonella sulcata was appeared in 29 animals with a total percentage of 24.16% (Table 1), all of these animals infected with Buxtonella sulcata cysts. These cysts are round or oval in shape, yellow in color reach 68.6-107.8 µm in diameter with a mean of 74.58 µm, these cysts surrounded by a two layered capsule (Fig.1). Four positive samples which infected with trophozoites (vegetative forms) with a percentage of 13.79% (Table 1). The vegetative forms were oval, with diameters of 107.8-137.2X 49-102.9, with a mean of 121.25X94.06 µm. The surface of the cilliate's cell was evenly covered with short cilia, deep syncystoma was at the anterior pole and the nucleus lies in the centre of the vegetative form (Fig.2). From Table (1) statistical analysis showed significant differences between calves and beef cattle and between calves and dairy cattle while no significant differences between beef and dairy cattle. From Table (2) it is evident that there were three degree of infection, low 51.72%, moderate 24.13% and high 24.13% according to the number of parasites under high power field (40x hpf) there was no significant differences between calves, beef and dairy cattle. Buxtonella sulcata cysts were appeared in 27 animals with diarrhea symptom in a percentage of 31.39% while those show no diarrhea, the percentage were 5.88% (Table 3). Statistical analysis showed significant differences between animals with and without diarrhea.

Materials and Methods Faecal samples were collected from 120 cattle (44 calves, 42 dairy frezinn cattle, and 34 beef cattle) from teaching veterinary Medicine College, farms of Agriculture College and Kogyali village and cattle market in Mosul city during October 2007 – April 2008, faecal samples were taken from animals with diarrheal disease (86) and apparently healthy animals (34), samples kept in a clean plastic container. Direct smear method (12), sedimentation methods by using ether-formalin (13,14) were used for detection Buxtonella sulcata. Differentiation of Buxtonella cyst or trophozoites was based on morphological specific feature and by microscopic measurement by using ocular micrometer (2,3,6,8,11). Coproscopical examination for detection the degree of intensity of infection according to (15,16) and examination of the 20 microscopical fields. The data were analyzed statistically by using Z-test, Fisher- test and χ2 – test (17).

Table (1): Incidence of Buxtonella sulcata of cattle in Mosul.

No. Of positive Rate of Infection with Infection with samples infection cyst No.(%) Trophozoite No.(%) Calves (3-8 months) 44 5 11.36 a 5(100.0) 0(0.0) Beef cattle (2-7 years) 34 12 35.29 b 12(100.0) 3(25.0) Dairy cattle (4-8 years) 42 12 28.57 b 12(100.0) 1(8.3) Total 120 29 24.16 29(100.0) 4(13.79) Rates with different letters have significant difference at P<0.05 according to Z-test between two proportion.

Animals

No. of samples

Table (2): Distribution and intensity of infection with Buxtonella sulcata of cattle.

Infection Animals No. of +ve samples Low degree* Moderate degree** High degree** Calves 5 4(80.0) 1(20.0) 0(0.0) Beef cattle 12 5(41.6) 3(25.0) 4(33.33) Dairy cattle 12 6(50.0) 3(25.0) 3(25.0) Total 29 15(51.72) 7(24.13) 7(24.13) Groups with same letters have non-significant difference at p<0.05 using Fisher Freeman Halton test.

Low degree: 1 cyst/hpf , Moderate degree: 2-4 cyst/hpf , High degree: 5 and more than 5 cyst/hpf

28

Statistical group A A A

Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (27-30)

Figure (1): Cysts of Buxtonella sulcata (10X,40X) by using digital camera.

Figure (2): Trophozoites of Buxtonella sulcata (10X,40X) by using digital camera.

Table (3): Relationship of Buxtonella sulcata infection with diarrheal symptoms in cattle by using Ď&#x2021;2 â&#x20AC;&#x201C; test.

Discussion

Symptoms

No. of animals

No.of +ve B.sulcata 27

In this study, the total percentage of infection with Buxtonella sulcata was 24.16% and the infection with cysts appeared in all positive cases while the trophozoites appeared only in four positive cases from total of all positive number, while other study accomplished in Iraq by Ayaiz (11) reported that the percentage of infection was 47%. Other studies which performed in different part of world such as England, Poland, Costa Rica, Bangalore and Thailand (2,6,9,18,19) showed a wide differences in percentage and ranged between 2-87%.The differences in the percentages of infection could be due to many different factors, such as environmental conditions, animal, farm management practices and stress factors. Fox and Jacobs (2) itself showed that seasonal fluctuations in the prevalence of the infection and cyst excretion rates were

% of infection

Animals with diarrhea 86 31.39a Animals with normal 34 2 5.88b faeces Total 120 29 24.16 Groups with different letters have significant differences at P<0.016.

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Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (27-30)

2. Fox MT, Jacobs DE. Pattern of infection with Buxtonella sulcata in British cattle. Res Vet Sci. 1986;41:90-92.

3. Rommel M, Eckert J, Kutzer E, Korting W, Schneider T. Veterinar Medizinische Parasitologie. Parey Buchverlag. Berlin 2000 [cited Tomczuk et al., 2005].

4. Vasily DB, Mitchell JB. The identification of rumen ciliates from eastern Bos. Taurus. Transaction of the American microscopical Society. 1974;93(2):248-253.

5. Becker ER, Hsiung TS. The methods by which ruminants acquire their Fauna of infusoria, and remarks concerning experiments of the host specificity of these protozoa. Zoology. 1929;15:684-690.

6. Tomczuk K, Kurek L, Stec A, Studzinska M, Mochol J. Incidence and clinical aspects of colon ciliate Buxtonella sulcata infection in cattle.

Bull Vet Inst Pulawy. 2005;49:29-33.

7. Henriksen SA. Buxtonella sulcata, an intestinal ciliate of apparently frequence occurrence in Danish cattle (author's transl). Nord Vet Med. 1977;29(10):452-7.

8. Hong KO, Youn HJ. Incidence of Buxtonella sulcata from cattle in Kyonggi-do. Korean J Parasitol. 1995;33(2):135-138.

9. Jimenez AE, Montenegro VM, Hernandez J, Dolz G, Maranda L, Galindo J, EPe C, Schineder T. Dynamics of infections with gastrointestinal parasites and Dictyocaulus viviparous in dairy and beef cattle from Costa Rica. Vet Parasitol. 2007;148:262-271.

10. Wackers K, Roffeis M, Conraths FJ. Cow-calf herds in eastern Germany: status Quo of some parasite species and a comparison of chemoprophylaxis and pasture management in the control of gastrointestinal nematodes. J Vet Med B. 1999;46(7):475-483.

11. Aayiz NN. Diagnostic study for cow infection with Buxtonella sulcata in Iraq. Al-Qadissiyha J Vet Sci. 2005;4(2):53-56.

12. Urquhart GM, Armour J, Duncan JL, Dunn AM, Jennings FW. Veterinary Parasitology. 2 nd ed. Black Well Science Ltd., Oxford; 2003.276p.

13. Mollan AA, Saaid ES. Principles of manual parasitology. University of Mosul; 1982.308p.

14. Thienpont D, Rochette F, Janparijs OFJ. Diagnostic helminthiasis through coprological examination. Beerse Belgium. 1979;34-36p.

15. Cox FEG. Modern Parasitology: A textbook of Parasitology. 2nd ed.

Black Well science; 1999:76-77p.

16. Bakir, MHH. The study of epidemiology experimental immunity and transmission for cryptosporidiosis in Nineveh governorate. [dissertation]. College of Veterinary Medicine: University of Mosul; 2005.40p.

17. Petrie A, Watson P. Statistics for veterinary and animal science. Black well science. London; 2003.

18. Mamatha GS, Pacid EDS. Gastrointestinal parasitism of cattle and buffaloes in and around Bangalore. J Vet Parasitol. 2006;20(2):846874.

19. Kaewthamasorn M, Wongsamee S. A preliminary survey of gastrointestinal and haemoparasites of beef cattle in the tropical live stock forming system in Nan Province, Northern Thailand.

Parasitology Research. 2006 (on line), short communication.

20. Nurialtug Y, Yuksek N, Ozkan C. Parasites detected in neonatal and young calves with diarrhoea. Bull Vet Inst Pulway. 2006;50:345-348.

21. Skotarczak B. Wplyw niekorych ezynnikow zewnetrznych na encystacje I ekscysacje Balantidium coli. Wind Parazytol. 1984;30: 566-573. [cited Tomczuk et al., 2005].

22. Skotarczak B, Zielinski R. Wplyw Wybranych Czynnikow biotopu Balantilium coli na przebieg balantidiozy swin. Wiad Parazytol. 1997; 43:399-403. [cited Tomczuk et al., 2005].

23. Urman HD, Kelky GD. Buxtonella sulcata. A ciliate associated with ulcerative colitis in a cow and prevalence of infection in Nebraska cattle. Iowa State Univ Vet. 1964;27:118-122.

24. Skotarczak B. Bacterial flora in acute symptom-free balantidiosis.

Acta Parasitol. 1997;42:230-233.

related to changes in the diet and opportunities for transmission, furthermore, the delivery rate may be lead to an increase in prevalence of infection. The shape and size of the cysts and trophozoites which observed in this study are in agreement with those described by (3, 8,11).

The statistical analysis of infection with Buxtonella sulcata in our study showed a significant differences between the infection of calves, beef and dairy cattle, while no significant differences was noticed between beef and dairy cattle, moreover, no significant differences were observed in the intensity of the infection between calves, beef and dairy cattle. High rate of infection with Buxtonala sulcata cysts appeared in animals affected with diarrhea and which are found to have large number of cysts and trophozoites (Table 3), the result was in agreement with (2,6,11,19,20) who reported that B. sulcata can be one of the causative agent of diarrhea in ruminants. The pathogenic effect of this parasite has not been found to be of great interest and it is suggested that more comprehensive studies should be done in order to explain the cause of diarrhea. In an observations of Tomczuk et al., (6) they reported that the pathogensis of B. sulcata may be compared with the incidence of similar ciliate Balantidium coli living in the large intestine of pig, man and many other mammals. In a different studies of Skotarczak and Zielinski, Urman and Kelky, and Skotarczak (21-24) was proved that especially significant effect of Balantidium coli in change in the pH of large intestine content on the intensively of invasion and the damaging effect on the mucosa of large intestine results in a secondary bacterial infection and increase in the pathological changes.

In this study, our observation was based on detection of the B. sulcata cysts in animals which have signs of diarrhea and those with normal faeces. All examined positive cases was affected with B. sulcata only, furthermore, no clinical signs observed other than diarrhea on the affected animals and no other parasitic agents were diagnosed, this indicates that B. sulcata can be considered as one of the agents of unexplained etiology of diarrhea in cattle.

Acknowledgement This study was supported by the College of Veterinary Medicine, University of Mosul.

References 1. Bauer C. Vorkommen and Beschreibung derzysten deszakumziliaten Buxtonella sulcata (Jameson,1926) im kot vomkuen in Norddentschland. Berl Munch Tieraztl Wschr. 1983,96:371-374. [cited Tomczuk et al., 2005].

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Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (31-35)

Clinical, haematological and biochemical studies of babesiosis in native goats in Mosul E. G. Sulaiman1, S. H. Arslan2, Q. T. Al-Obaidi2, E. Daham2 1

Department of Microbiology, 2Department of Internal and Preventive Medicine, College of Veterinary Medicine, University of Mosul, Mosul, Iraq (Received March 26, 2008; Accepted July 13, 2009)

Abstract The study included examination of 175 native goats, 27 were infected with Babesia ovis, B. motasi, B. foliata and B. taylori, (recorded in Mosul for the first time) and 25 were clinically normal and served as control. Results indicated that the percentage of the infection with Babesiosis was 15.42% and the percentage of parasitemia ranged between 3.5-10.4% with a mean 6.95%, infected goats showed signs of loss of appetite, weakness, pale mucous membranes, jaundice, fever, coughing, nasal discharge, recumbency, diarrhea and haemoglobinuria. A statistically significant decrease were recorded in total red blood cells (RBC), haemoglobin concentration (Hb), packed cell volume (PCV) and platelets counts. Anemia was of microcytic hypochromic type. A statistically significant increase in erythrocyte sedimentation rate and significant increase in total white blood cells was recorded due to significant increase in lymphocyte and neutrophile count. Results of the biochemical testes indicated an increase in activity of alanin amino transferase (AST), aspartate amino transferase (ALT), total bilirubin, blood urea nitrogen and icterus index, with a significant decrease in total serum protein, albumin and globulin levels.

Results also indicated the presence of Rhipecephalus ticks which were: Rh. sanguineus and Rh. turanicus.

Keywords: Babesia, Goats, Hematology.

Available online at http://www.vetmedmosul.org/ijvs

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31

Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (31-35)

Blood was collected from jugular vein for haematological examination by using (Automatic full digital cell counter, Beckman USA) to get of total red blood cells (RBC), haemoglobin concentration (Hb), packed cell volume (PCV), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), platelets counts and total and differential leuckoytes count (TLC & DLC), and westergren method used for get erythrocyte sedimentation rate (ESR) (17). Thin blood smears were taken from the vein of the ear and stained with Giemsa were used to identify the Babesia and the percentage of parasitemia (18). Blood serum samples were tested spectrophotometrically for the biochemical changes of alanine amino transferase, aspartate amino transferase, blood urea nitrogen and total serum protein, albumin and globulin by using available kits (Randox, U.K.), total bilirubine using available kits (Biomerex, France), and Icterus Index by using potassium dichromate according to (18). Statistical analysis were done by using t-test (19).

Introduction Babesiosis is caused by intraerythrocytic protozoan parasites of the genus Babesia, the disease which is transmitted by hard ticks (family: Ixodidae), affect a wide range of domestic and wild animals and occasionally humans (1). Babesia occurs seasonally and the peaks of infection were observed in rainy season (2). Babesia forms can vary as pear-shaped, round and elongated (3) Clinically Babesiosis is characterized by fever, inappetence, increased respiratory rate, muscle tremors, anemia, jaundice, body weight loss, and hemoglobinuria in the final stages (4,5).

Anemia is very common for all infected animals, hemoglobinuria may not observed in animals infected with B.ovis (6) Four species of Babesia have been reported from sheep and goats mainly consisting of one large form (B. motasi) and three small forms (B. ovis, B. foliata and B. tyalori) (7), while Friedhoff referred that the Babesiosis in domestic small ruminants is due to at least three species, namely : B. ovis, B. motasi and B. crassa (8). B. ovis is less pathogenic than B. motasi for sheep infection and cause relatively moderate haemolytic anemia (9,10), whereas Friedhoff considered the B. ovis is the most important causative agent which transmitted by Rhipicephalus bursa, R. turanicus, Hyalomma anatolicum excavatum and probably by R. evertsi evertsi (11), whereas the known vector of B. motasi are Haemophysalis punctata and R. bursa and the B. motasi is more pathogenic than B. ovis in India and northern Africa. In animls affected with Babesia spp. The studies revealed that decrease in the total erythrocytes counts, haemoglobin concentration,packed cell volume and platelets counts, and in biochemicals studies showed increase in activity of alanine amino transferase, aspartate amino transferase, total bilirubin, blood urea nitrogen and icterus index, with decrease in total serum protein. (12-14) The percentage of infection with B. ovis in goats in AlArich city and El-Hassanah center were 7.0% (15) whereas the prevalence of B. ovis infection in Awassi sheep in Urfa, Turkey was 41.02% (16). Little work has been done on Babesia spp in Mosul, Iraq on goats, hence the present study was taken to determine the occurrence of Babesia spp together with clinical, haematological and some biochemical changes.

Results Clinically infected goats showed different signs graduated from loss of appetite, emaciation, pale mucous membranes, jaundice, fever, coughing, nasal discharge, recumbency, diarrhea and haemoglobinuria as well as presence of ticks (Rhipicephalus sanguineus and Rh. turanicus) were detected on different parts of the body (Table 1).

Table (1): Clinical signs of infected goats (n=27) with babesiosis.

Clinical signs Pale mucous membranes Loss of appetite Haemoglobinuria Diarrhea Emaciation Jaundice Nasal discharge Coughing Recumbency Ticks (Rhipicephalus sanguineus and Rh. turanicus)

Materials and methods A total of 175 goats, 2-5 years of age and from both sexes were examined in the Teaching Veterinary Hospital (College of Veterinary Medicine, University of Mosul) and from other regions in Mosul city, for the possibility of infection with babesiosis. Clinically normal goats (n=25) served as control. Careful clinical examination of all suspected animal were carried out.

No. of infected goats 24 21 21 16 15 13 10 9 7

88.9 77.8 77.8 59.3 55.6 48.1 37 33.3 25.9

15

55.6

%

Significant increase (P<0.01) were encountered in body temperature, respiratory and heart rates While, ruminal contractions were reduced significantly (Table 2).

Examination of blood smear revealed four types of Babesia in infected goats, which were: B.ovis,, B.taylori, B.foliata and B. motasi. (Fig1). The number of goats which

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Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (31-35)

were infected with Babesia spp was (27) with percentage (15.42%) and the parasitemia ranged between (3.5-10.4%) in a mean of (6.95%).

Table (2): Body temperature, respiratory rates, heart rates and ruminal contractions of infected goats with babesiosis.

Parameters Body temperature oC Respiratory rate/min Heart rate/min Ruminal contraction/5 min ** P<0.01, * P<0.05

B. ovis (round form) at the margin of the red cells (1.66-2.32) micron with mean 1.88 micron (1).

Control Mean±S.E. 39.10±0.51 26.21±3.82 79.81±8.62 4.09±0.20

Infected Mean±S.E. 40.8±0.82** 53.32±7.95** 112.4±10.90** 2.52±1.5**

There was a significant reduction (P<0.01) in the mean values of TRBC, Hb, PCV, platelets count, anemia was of Microcytic hypochromic type due to significant reduction (P<0.01) in the Mean Corpuscular Volume (MCV) and Mean Corpuscular Haemoglubin Concentration (MCHC).

A statistically significant increase of ESR encountered and results also indicated a significant increase (P<0.01) in total leukocytes count due to significant increase in neutrophils and lymphocytes count (Table 3).

B.taylori (round appear) under go several fission (1.661.19) micron with mean 1.74 micron (2) .

Table (3): Blood parameters of infected goats with babesiosis and control group.

Parameters RBC x 106 microliter Hb g/100 ml PCV % MCV % MCHC g/100 ml Platelets x 103 microliter ESR mm/24 h WBC x 103 microliter Neutrophils % Lymphocytes % Monocytes % Eosinophils % Basophils % Parasitemia ** P<0.01, * P<0.05.

B. foliata. round lies more centrally in red cells (1.66-1.99) micron with mean 1.83 micron (3).

B.motasi (pyriform stages) (2.94.15) micron with mean 3.27 micron (4) .

Control Mean ± S.E. 6.10 ± 0.54 10.35 ± 2.43 32.40 ± 3.22 64.37 ± 5.68 32.35 ± 4.33 769 ± 157 0.62 ± 0.43 7.68 ± 2.35 61.33 ± 1.46 37.50 ± 0.93 0.20 ± 0.03 0.15 ± 0.03 0.04 ± 0.01 ---

Infected Mean ± S.E. 4.21 ± 1.15** 6.93 ± 2.52** 26.13 ± 4.33** 61.40 ± 5.46** 28.31 ± 2.20** 349 ± 103** 3.94 ± 1.34** 10.95 ± 3.73** 61.87 ± 1.02* 39.20 ± 1.31** 0.15 ± 0.10 0.00 ± 0.00 0.06 ± 0.01 3.5 – 10.4%

Results of biochemical examination showed a significant increase (P<0.01) in activity of AST, ALT, as well as in levels of total bilirubin, BUN and Icterus index, however significant decrease were recorded in total protein, albumin and globuline values of infected goats (Table 4).

Figure (1): four types of babesia in infected goats, which were: B.ovis (1), B.taylori (2), B.foliata (3) and B. motas i(4) in blood smear.

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Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (31-35)

Anemia was microcytic hypochromic type due to significant reduction in the mean corpuscular volume and mean corpuscular haemoglubin concentration this was in agreement with the study of (23) in foals. The increase in ESR values refers to the correlation between the sedimentation of RBCs and the intensity of anemia (18), and Allen added that increase in ESR values due to decrease in Packed cell volume (24). The significant increase in WBCs was due to increase in lymphocytes, this was in agreement with (3) in sheep.

Biochemical parameters showed relatively significant increase in the AST and ALT may be due to indirect damage of liver, kidney tissue and myocardium, changes indicated to a possible damage to the liver, kidney tissue (25), Wright added that increase in the AST due to distraction of RBCs (26). ` The increase in total bilirubin due to damage of liver and increase in the indirect bilirubin was due to erythrocyte haemolysis (18), Babesia also reported to causes nephrosis, renal ischemia, dehydration and some heart diseases that causes increase in BUN, (26,27). The increase in the icterus index was due to increase in Total bilirubin in serum and dehydration (26).

The reduction in total protein, albumin and glubulin values is might be due to decrease production from the liver due to direct and indirect effect of parasite, digestive distribance (diarrhea), loss of appetite and high fever (28,29).

Table (4): Biochemical parameters of infected goats with babesiosis and control group.

Control Mean ± S.E. AST U/L 27.13 ± 9.52 ALT U/L 23.65 ± 5.12 Total bilirubin mg/100 ml 0.26 ± 0.10 Total protein g/100 ml 6.70 ± 0.79 albumin g/100 ml 4.07 ± 0.22 Globulin g/100 ml 2.64 ± 0.57 BUN mg/100 ml 32.78 ± 7.64 Icterus index units 2.67 ± 0.39 ** P<0.01, * P<0.05.

Parameters

Infected Mean ± S.E. 77.37 ± 6.94** 29.63 ± 4.59** 0.69 ± 0.17** 3.63 ± 0.53** 2.10 ± 0.21** 1.27 ± 0.12* 68.46 ± 12.89** 6.31 ± 1.16**

Discussion The results of this study showed that the clinical signs observed in infected goats were in agreement with the results of other studies carried out by different researchers concerning ruminants infected with babesiosis (2,3,7). The cause of pale mucous membrane was development of anemia and decrease in erythrocyte count and haemoglobin (12), while jaundice due to increase in the total bilirubine (direct and indirect) and icterus index (18), the haemoglobinuria may be due to intravascular haemolysis and high rate of destruction of erythrocytes and haemoglobinemia (20,21).

Two types of ticks were diagnosed in this study, they were Rhipicephalus sanguninus and Rh. turanicus and Friedhoff added that Babesia ovis is transmitted by Rh. bursa, Rh. turanicus Hyalomma anatolicum excavatun and probably R. evertsi evertsi (11), while Mazyed and Khalaf identified Hyalomma anatolicum excavatum and Haemophysalis sulcata in infected goats with B. ovis and Theileria ovis (15). In general the distribution of the parasite is correlated with the distribution of tick vector species (16). Four species of babesia (B. ovis, B. motasi, B. foliata, B. taylori) were diagnosed in this study, which were also described by (7,12).

The percentage of infection with Babesia spp was (15.42%) whereas Mazyed and Khalaf recorded (7%) of infection with B. ovis in goats (15) and in another study on goats in a village in south west Nigeria showed (20.4%) infection with B. motasi (2).

Haematological parameters showed relatively significant decrease in values total Red blood cells, haemoglobin concentration, packed cell volume and platelets counts, compared to control group, these results were in agreement with studies carried out on sheep and goats by others (2,12,22) and this might be due to intravascular haemolysis of erythrocyte, increase erythrocyte phagocytosis by reticloendothelial system and restricted erythropoitic activity in bone marrow (20,21).

Acknowledgement This study was supported by the College of Veterinary Medicine, University of Mosul.

References 1. Merck Co. The Merck veterinary manual. Babesiosis: Overview, published in educational partner ship, USA, 2006. Available from: http://www.merk.vetmanual.com/mvm/htm/bc/10402.htm.

2. Opasine BA. Blood parasites of village goats in South West Nigeria.

Animal Genetic Res. 1984.

3. Kozat S, Yuksek N, Altug N, Agaoglu ZT, Ercin F. Studies on the effect of Iron (Fe) Preparations in addition to Babesiosis treatment on the haematological and some mineral levels in sheep naturally infected with Babesia ovis, YYU Vet. Fak Derg. 2003;14(2):18-21.

4. Urquhart GM, Armour J, Duncan JL, Dunn AM, Jennings FW. Veterinary parasitology. 2nd ed. Blackwell Co. Great Briatain; 1996.

242-245p.

5. Ferrer D, Castella J, Guticrrez JF, Lavin S, Marco I. Seroprevalence of Babesia ovis in Mouflon sheep in Spain. J Wildlife diseases. 1998; 34(3):637-639.

6. Popa E. Ixodid ticks vectors of Babesiosis in animals in Romania.

Revista-Romana-de-Medicina-Veterinara. 1998;8(2):61-67.

7. Soulsby EJL. Helminths, Arthopods and Protozoa of domesticated animals. 7th ed. Philadelphia, London, Toronto. 1986.718-719p.

8. Friedhoff KT. Transmission of Babesia, In: Babesiosis of domestic animals and man. M. Ristic(ed.) CRC. Press, Boca Raton. Florida.

1988.23-52p.

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9. Aytug CN, Alacam E, Gorgul S, Koyun Ve. Keci Hastaliklarive Yetistiri cillige Istanbul, Tum-Vet Hayvancilik Yayini. 1990;201-202.

10. Imren HY, Sahal M. Veteriner ic Hastaliklari. Ankara. Feryal Matbacilik San. ve Tic. Ltd. Sti. 1991;207-208p.

11. Friedhoff KT. Tick-borne diseases of sheep and goats caused by Babesia, Theileria or Anaplasma spp. Parasitologia. 1997;39(2):99109.

12. Radostitis OM, Gay CC, Blood DC, Hinchcliff. Veterinary medicine, A text of the diseases of cattle, sheep, pigs, goats and horse. 9th ed.

Philadelphia: W.B. Saunders Company; 2000.1289-1296p.

13. Arsalan SH. Clinical, haematological and biochemical studies of some blood protozoa in dogs in ninavah. Iraqi J Vet Sci. 2005;1(19):63-77.

14. Al-Mula GM. Clinical, pathological and therapeutical studies of Equine Babesiosis in draught horses in mosul. [master's thesis].

Collage of Veterinary Medicine: University of Mosul; 2004.

15. Mazyed SA, Khalaf SA. Studies on Theileria and Babesia infecting live and slaughtered animals in Al-Arish and El-Hassnah, North Sinai Governorate, Egypt. J Egypt Soc Parasitol. 2002;32(2):601-610.

16. Emre Z, Duzgun A, Iriadam M, Sert H. Seroprevalence of Babesia ovis in Awassi sheep in Urfa, Turkey. Turk J Vet Anim Sci. 2001;25: 759-762.

17. Meyer DJ, Harvy JW. Veterinary laboratory medicine. 2nd ed. W.B. Saunders Co; 1998.157-199p.

18. Jain NC. Schalm's Veterinary hematology. 4th ed. Lea and Febiger, Philadelphia; 1986.610-612p.

19. Steel RG, Torrie JH. Principles and Procedures of Statistics. 2nd ed.

McGraw, Hill Inc.; 1985.120p.

20. Lewis D, Holman MR, Purnell RE, Young ER, Herbert IV, Bevar WJ. Investigation on Babesia motasi isolated from wales. Res Vet Sci. 1981;31(2):239-243.

21. Voyvoda H, Sekin S, Kaya A, Bildik A. Koyunlarin Dogal Babesia ovis Enfeksiyonunda Serum Demir. Bakir Konsantrasyona (Fe, Cu) total Ve Latent Demir Baglama Kapasitesi (TDBK : LDBK) Ve Transferrin Doynma (TD) Modifikasyonlari Tr. J Vet Ani Sci. 1997;21(1):31-37.

22. Alani AJ, Herbert IV. The pathogenesis of Babesia motasi (Wales) Infection in sheep. Vet Parasitol. 1998;27:202-220.

23. Guimaraes AM, Lima JD, Tafuri WL, Ribeiro MF, Sciavicco CJ, Botelho AC. Clinical and histopathological aspects of splenectomized foals infected by Babesia equi. JEVS.1997;17(4):211-216.

24. Allen BV. Relationships between the erythrocyte sedimentation rate, plasma proteins and viscosity, and leukocytes counts in thoroughbred racehorses. Vet Rec. 1988;122:329-332.

25. Yfruham I, Hadani A, Galker F, Avidar Y, Bogin E. Some epizootiological and clinical aspects of ovine babesiosis caused by Babesia ovis. Vet Parasitol. 1998;4(2-4):153-63.

26. Wright IG. Biochemical characteristics of Babesia and physiochemical reactions in host. In: Babesiosis, ed. By: Ristic M, Kreier JP. Academic press, New York & London; 171-205p.

27. Coles EH. Veterinary clinical pathology. 4th ed. Philadelphia: W.B. Saunders Co; 1984.25-29p.

28. Al-Aboud AY, Al-Deoun MA, Maroun EA. Haematological and histopathological in sheep and goats naturally infected with some single blood protozoa. Bas J Vet Res. 2005;4(1):10-14.

29. Hailat NQ, Lafi SQ, Al-Darraj AM, Al-Ani FK. Equine Babesiosis associated with strenuous execise: clinical and pathological studies in Jordon. Vet Parasitol. 1997;69:1-8.

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Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (37-40)

Effect of industrial product IMBOÂŽ on immunosuppressed broilers vaccinated with Newcastle disease vaccine O. G. Mohammadamin* and T. S. Qubih Department of Pathology and Poultry Diseases, College of Veterinary Medicine, University of Mosul, Mosul, Iraq (Received May 13, 2009; Accepted October 5, 2009) Abstract The effect of IMBO was investigated on humoral immune response to Newcastle disease vaccines in broiler chickens.

Haemagglutination inhibition test and enzyme-linked immunosorbent assay were used to assess the immune response. Results showed that although IMBO significantly enhanced humoral immune response to live Newcastle disease vaccine, it did not decrease post virulent NDV challenge mortality.

Keywords: Humoral immunity; Newcastle disease,Vaccine, IMBO. Available online at http://www.vetmedmosul.org/ijvs

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and increased macrophage phagocytic ability which positively correlates with enhanced resistance to various viral and bacterial infections (8,9). Probiotics are defined as direct feed microbials or microbial cell preparations with a beneficial effect on the health and well-being of the host (10). Probiotic represent one of the most recent examples of natural substances that influence adaptive immune responses by activating the innate immune system (11), and enhancing the systemic antibody.response to some antigens in chickens (12). Recently, the beneficail effect of BiominÂŽ C-X (Enterotococcus faecium + prebiotic+cell wall extract) on humoral immunity to Newcastle disease vaccine of commercial broilers was studied (13). This experiment was conductd to investigate the effect of BiominÂŽIMBO (Biomin G.T.I. GmbH., Ember AG-Austria; containing Enterotococcus faecium 5Ă&#x2014;1011 cfu /kg, prebiotic, cell wall

Introduction Severe outbreaks of Newcastle disease often occur in areas of° intensive poultry production, which is reasoned mainly to break down in immunity. Although poor vaccine quality is one of several possible factors that could lead to vaccination failures (1).The failure of protection usually results from: (i) mycotoxin and/or drug induced immunomodulation (ii) cold or heat stress (iii) infectious agents (iv) malfunctioning of the host defense mechanism and (v) presence of high titers of maternal antibodies (1-7).

Immunostimulation of a bird may lead to increased antibody production, increased cellular immune responses, *

Part of MSc thesis submitted by the first auther to the College of veterinary Medicine, Mosul University.

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Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (37-40)

and algae extracts) as a potential immunostimulator to enhance humoral immune response to live and killed Newcastle disease vaccines in broiler chickens.

CPA + Non challenge referred to as negative control (G1), 2) IMBO + CPA&L-K (G2), 3) IMBO + Non CPA&L (G3), 4) IMBO + Non CPA&L-K (G4), 5) Non IMBO + CPA&L-K (G5), 6) Non IMBO + Non CPA&L (G6), 7) Non IMBO + Non CPA&L-K (G7). Birds of all groups except negative and positive controls were vaccinated with live NDV (Cevac®Vitapest L; CEVA) at seven days old individually by oral route using 1 ml syringe. In addition, each bird in groups G2,G4, G5, and G7 was intramuscularly injected with 0.1 ml of killed NDV vaccine (Cevac®Broiler NDK) at seven days of age. Revaccination with live ND vaccine LaSota strain (Cevac® NEW L;CEVA) was done at 21 days of age by spraying.

Materials and methods A total of 210 day-old Hubbard-Flex broiler chicks were procured from a local supplier. They were reared in cages in a separate rooms of the animal house, College of Veterinary Medicine, University of Mosul and fed ad libitum with a Hubbard-Flex recommended diet. Ambient temperature, lighting, ventilation and other environmental conditions fully met the requirements for management of Hubbard-Flex birds.

Results Biomin®IMBO (Biomin G.T.I. GmbH., Ember AG-Austria, it contains Enterotococcus faecium 5×1011 cfu / kg, prebiotic, cell wall and algae extracts. IMBO was added to the feed free from antibiotics and administered throughout the study as recommended by the manufacturer 1.5g/kg.

HI titer serum antibody response According to figure (1), chicks contained maternal antibody level before vaccination and gradually declined to low levels with time. At 28 days of age, production of antibody detected in all groups except G1 group. The G2 (vaccine + IMBO+CPA) and G5 (vaccine + CPA) groups produced significantly (P<0.05) lower levels of antibody in comparison to other treatment groups indicating that immunosuppression occurred. Furthermore, no significant differences were found when the two groups were compared at different time points post booster vaccination.

The data also revealed that GMT of G3 ( live vaccine + IMBO) group was significantly (P<0.05) higher than G6 (live vaccine alone) group while GMT of G4 (live& killed vaccine + IMBO) group was statistically (P>0.05) not different compared with G7 (live& killed vaccines).

Furthermore, when GMTs of G3 and G4 groups were compared no significant (P>0.05) difference were found.

Drugs Cyclophosphamide (CPA) (Cycloxan® manufaured in India, Biochem Pharmaceutical Industries LTD) was procured from a local pharmacy. Day-old chickens of groups G2 and G5 were given 3 mg per chicken per day for 4 consecutive days intramuscularly into leg muscle (14).

Challenge virus One day before challenge, birds in G1 splited randomely to two halves; negative control group (G1); left wthout challeneg and positive control group (G8) which submitted to challenge. At 39 days of age chickens were intramuscularly inoculated with virulent field NDV strain (obtained from the Microbiology Department, College of Veterinary Medicine, Mosul University). The virus titer was determined to be 1×106.5 EID50 / 0.1ml.

ELISA titer immune response The results of ELISA test are presented in figure (2). On day 28, only birds in G3 and G7 groups showed seroconversions, however their mean titers were not significantly (P>0.05) different. The data also demonstrated that the antibody titer of G3 was significantly higher (P<0.05) when compared with titer of G6, meanwhile the titer of G4 was not significantly (P>0.05) different when compared with G7. The titers of G2 and G5 groups remained low and did not differ signficantly.

Sampling On day 7(before vaccination 0, blood samples were taken from each group to assess the maternal immunity.

Blood samples were taken at weekly intervals after vaccination and challenge.

Serological Test Antibodies to NDV were quantitified by hemagglunation inhibition test (HI) using the diluted serum-constant virus procedure (15) and by indirect Enzyme-linked Immunosorbent Assay (ELISA).

Mortality The post-challenge test results are shown in Table (1).

The table shows that the protection rate in G2, G3,G4, G5, G6, G7 and G8 were 28.6, 85.71%, 96.29%, 31.8%, 83.87, 96.66%, and 0% respectively. No significant differences in mortality were found between probiotic fed and their corresponding control groups.

Experimental design Chickens were randomly divided to 7 groups with 30 birds each. These groups consisted of: 1) Non IMBO + Non

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Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (37-40)

Discussion In this experiment, significantly higher HI and ELISA titers were seen in birds received live NDV +IMBO(G3).

This is in agreement with finding of (13), however, IMBO had no effect in birds immunized with live and killed vaccine G4. The data on the effect of probiotics on immunity are extremely controversial due to the variety of variables reported (16). More over(17) reported treating with just one bacterial type may not be as effective and increasing the types of bacteria in the mix could enhance the efficacy of probiotic functions. The post-challenge mortality rates observed in immunosuppressed (G2 and G5 groups) were higher compared with immune-competent birds (G3, G4, G6 and G7 groups). The protection rate did not differ significantly in G3 compared with G6 despite enhancement of humoral immune response which contradicts preivious report (18). The different results might be due to twofold increase in titer observed in latter study and in addition, they challenged birds orally with virulent NDV compared with IM challenge used in our study, in addition Leghorn male chickens were used compared to broilers in the present experiment. Under the conditions of this study, IMBO significantly enhaced humoral immune response to only live vaccine,and this is in agreement with (19), but did not restore immunity in immunosuppressed chickens and did not decrease post challenge mortality in immunosuppressed and immunocompetent broiler chickens.

Fig. 1: Geometric mean HI antibody titer (log2) in chickens with or without IMBO supplementation.

Fig. 2: Mean ELISA antibody titer in chickens with or without IMBO supplementation.

References

Table 1: Response to challenge with virulent Newcastle disease virus in chickens

Groups Negative control group (G1) IMBO,CPA &L-K vaccine (G2) IMBO&live vaccine (G3) IMBO&L-K vaccine (G4) CPA &L-K vaccine (G5) Live vaccine (G6) L-K vaccine (G7) Positive control group G8)

No dead/No challenged 0/14 20/28 4/28 1/27 15/22 5/31 1/30 13/13

1. Chaudhry K M, Chaudhry RA. Causes of vaccine failure. Vet Int. 1996;12: 13-15 2. Li Y C, Ledoux D R, Bermudez A J, Fritsche K L, Rottinghaus G E. Effects of fumonisin B1 on selected immune responses in broiler chicks. Poult Sci. 1999;78:1275-1282.

3. Sharma J M, Kim I J, Rautenschlein S, Yeh H Y. Infectious bursal disease virus of chickens: pathogenesis and immunosuppression. Dev Comp Immunol. 2000;24:223-235.

4. El-Lethey H, Huber-Eicher B, Jungi T W. Exploration of stressinduced immunosuppression in chickens reveals both stress- resistant and stress-susceptible antigen responses. Vet Immunol Immunopathol.

2003;95:91-101 5. Shivachandra SB, Sah RL, Singh SD, Kataria J M, Manimaran K. Immunosuppression in broiler chicks fed aflatoxin and inoculated with fowl adenovirus serotype-4 (FAV-4) associated with hydropericardium syndrome. Vet Res Com. 2003;27:39-51 6. Kim Y, Brown TP, Pantin-Jackwood MJ. The effects of cyclophosphamide treatment on the pathogenesis of subgroup J avian leukosis virus (ALV-J) infection in broiler chickens with Marekâ&#x20AC;&#x2122;s disease virus exposure. JVet Sci. 2004;5:49-58.

7. Muruganandan S, Lai J, Gupta PK. Immunotherapeutic effects of mangiferin mediated by the inhibition of oxidative stress to activated lymphocytes, neutrophils and macrophages. Toxicology. 2005; 215: 57-68.

8. Dugas B, Mercenier A, Lenoir-Wijnkoop I, Arnaud C. Dugas N and Postaire E. Immunity and probiotics. Immunol Today. 1999;20:387390.

No survived/No challenged 14/14 a (100)B 8/28 c (28.57) 24/28 a (85.71) 26/27 a (96.29) 7/22 c (31.81) 26/31 a (83.87) 29/30 a (96.66) 0b

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Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (37-40)

9. Maassen C B J D Laman W J Boersma and E Claassen. Modulation of cytokine expression by lactobacilli and its possible therapeutic use. In R. Fuller and G. Perdigon (ed.), Probiotics2001; 3: immunomodulation 10. Nemcovรก R. Criteria for selection of lactobacilli for probiotic use. Vet. Med. 1997; 42: 19-27.

11. Maldonado Galdeano C and Perdigo G. The probiotic bacterium Lactobacillus casei induces activation of the gut mucosal immune system through innate immunity. Clin. vaccine Immunol. 2006; 13: 219-226.

12. Haghighi H R Gong J Gyles C L Hayes M A Sanei B Parvizi P Gisavi H Chambers J R and Sharif S H. Modulation of antibody-mediated immune response by probiotics in chickens. Clin. Diagn. Lab. Immunol. 12:1387-1392. by the gut microflora and probiotics. Kluwer Academic, Dordrecht, the Netherlands. 2005; PP: 176-192.

13. Al-Dalo N R A A. Effects of probiotics on some aspects of pathology and productivity of chicken broilers. A Ph.D. Thesis. College of Veterinary Medicine, University of Mosul. Mosul. Iraq. 2007

14. Sharma J M and Lee L F. Suppressive effect of cyclophosphamide on the T-cell system in chickens. Infect. Immun. 1977; 17:227-230.

15. Young M Alders R Grimes S Spradbrow P Dias P da Silva A and LoboQ. Controlling Newcastle disease in Village Chickens: A laboratory manual.PP:72-85.2002 16. De Roos N M and Katan M B. Effects of probiotic bacteria on diarrhea lipid metabolism and carcinogenesis: A review of papers published between 1988 and 1998. Am. J. Clin. Nutr. 2000 71:405411.

17. Sharif S. Probiotics Help Produce Safer, Healthier Chickens.

University of Guelph Ontario Veterinary College. College News Published January 19. 2007; 338 Views 18. Zhang X Zhang X and Yang Q. Effect of compound mucosal immune adjuvant on mucosal and systemic immune responses in chicken orally vaccinated with attenuated Newcastle-disease.

Vaccine.

2007;25:3254-3262.

19. Emad J K and Amjad H E.Study of some Impact of Enterococcus faecium As Probiotic on chick 2-NewCastle Disease Antibody and White blood cells.Iraqi J Vet Med 2007;31(1):78-86.

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Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (41-43)

Serodiagnosis of Johne's disease by indirect ELISA in ovine I. M. Ahmed Department of Microbiology, College of Veterinary Medicine, University of Mosul, Mosul, Iraq (Received August 11, 2008; Accepted October 5, 2009) Abstract The study included collection of 92 serum samples from local Awasii breed in Mosul and Karakosh regions, some of them show clinical signs for John's disease, all samples were assayed using indirect Enzyme-Linked Immunosorbent Assay (ELISA) to detect antibodies against Mycobacterium avium subsp. paratuberculosis (Map). The results showed that 7/92 (7.6%) samples were positive for antibodies against (Map), and 7/89 (7.9%) were positive in female and 0/3 (0%) in male.

Keywords: Mycobacterium, Paratuberculosis, Ovine Johneâ&#x20AC;&#x2122;s disease, Indirect ELISA, Serodiagnosis.

Available online at http://www.vetmedmosul.org/ijvs

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.%E subclinical infections is difficult. Animals apparently are infected when young but, while shedding the organism via faeces, these animals may not show clinical symptoms for several years. Infected animals may have reduced feed efficiency without obvious clinical signs of disease. (4,5).

In sheep flocks, the fecal culture detects less than 12 % of clinical cases and requires up to 12 months of incubation, making it an impractical diagnostic test (6,7). Three different tests are currently available for measuring antibodies against Mycobacterium avium subsp. paratuberculosis in the serum of infected animals. These are the complement fixation test (CFT), the agar gel immunodiffusion (AGID) test and enzyme-linked immunosorbent assay (ELISA). ELISA or AGID are still the main options in live animals (8). Among various

Introduction Johne's disease is also known as "paratuberculosis" because it is caused by Mycobacterium avium subspecies paratuberculosis (Map). It is a contagious disease in dairy cattle, sheep, goats and other ruminants (1). The clinical manifestation of paratuberculosis in sheep tends to prevail at younger age than in cattle. Chronic weight loss is the primary clinical sign of paratuberculosis in sheep and goats.

Affected sheep will experience progressive weight loss over a period of weeks to months and eventually die (2). Only 10-20% of clinical cases present with diarrhoea or clumping of faeces in the advanced stage of the disease (3).

Johneâ&#x20AC;&#x2122;s disease is commonly found in dairy cattle herds and sheep flocks but identification of individual sheep with

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Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (41-43)

serological tests for Johne’s disease, ELISA-based tests are widely used and can be conducted rapidly and require limited expertise (9).

Materials and methods A total of 92 blood samples, 3 samples from males and the others from females were collected from local Awassi breed sheep that some of them showed clinical signs for John's disease including emaciation, unresponsive to dewormers and antibiotics. Appetite was often good, in spite of weight loss, all animals wer not previously vaccinated against Johne's disease and they were >2 years old. The samples were collected through April 2008 from different regions, 69 samples represent 3 flocks containing 580 sheep from Mosul and 23 samples represent 2 flocks containing 264 sheep from Karakosh. Samples were submitted to the department of microbiology (College of Veterinary Medicine, Mosul, Iraq); within 24 h after bleeding, serum samples were separated and stored at – 20°C until they were assayed (2,10).

A commercial ELISA kit (ID SCREEN®, Paratuberculosis Indirect) for detection of antibodies against Mycobacterium avium subsp. paratuberculosis in ovine serum samples was used, the kit has been supplied from (ID Vet (innovative diagnostics)-France). The principle of the test depends on indirect ELISA. Sera were tested according to the manufacturer's instructions for ovine, the absorbance reading O.D in all ELISA plate wells were measured at 450 nm using an automated ELISA reader. ELISA optical density (OD) readings were transformed to Serum/Positive percentage (S/P%) according to a specific equation cited by manufacturer. The sample considered positive if it gives S/P % ≥ 70%, 60% < S/P % < 70% considered doubtful, S/P % ≤ 60% considered negative. S/P%=(OD sample-ODNC)/(ODPC-ODNC).

Figure 1: Distribution of ELISA S/P % values for (n=92), positive controls (PC) and negative controls (NC). ID SCREEN®, Paratuberculosis Indirect ELISA kit, ID Vet (innovative diagnostics).

Discussion The aim of this study was to investigate Johne's disease in sheep by serodiagnosis using ELISA as there are indications for M. avium subsp. paratuberculosis infection and unresponsive to dewormers and antibiotics. All sheep were selected >2 years old as the clinical signs are commonly not evident until at least 18 months of age (6).

As the cultivation of sheep strains of M. avium subsp. paratuberculosis using culture media (Herrold's egg yolk medium) has been extremely difficult to perform, the selection of ELISA in this study was based on the studies by (6,12,13). Among the antibody tests, ELISA is more sensitive than AGID and CFT test (14,15). It's performance is similar in cattle, sheep, and goats (16,17) and can be used with comparable sensitivity for either milk or serum samples (18). AGID test is considered less sensitive than ELISA and CFT (19). Since a strong humoral response does not occur until the later stage of Johne's disease, the sensitivity of these 3 tests is the highest for animals with lepromatous lesions (8,20,21), those with clinical symptoms (20,22,23), or those that shed large numbers of bacteria (18,24). Therefore, the main limitation of these antibody tests is their inability to identify animals in early infection (25,26). Conversely, all of these tests are highly specific, with false-positive results occurring at low frequency (25).

The ELISA couldn't detect all animals with clinical signs.

Comparative studies of the CFT, AGID test and ELISA repeatedly show discrepancies in the ability to identify all infected animals (27,28). This may be due to genetic variation of the individual animal or the lack of representation of the entire range of immunodominant antigens for Mycobacterium avium subsp. paratuberculosis within a given test (29). There are few studies about ovine Johne's disease in the Arab countries, so there is no

Results The results showed that 7/92 samples 7.6% were positive for antibodies against Mycobacterium avium subsp. paratuberculosis, P<0.001. The results are detailed in table 1. Also the results showed no positive results in males 0/3 (0.0%) when compared with females 7/89 (7.9%). The distribution of S/P % values for samples, positive control and negative control are given in figure 1.

Table 1: Positive samples according to Mosul regions Region

No. of samples

Positive

%

Mosul Karakosh Total

69 23 92

5 2 7

7.3 8.7 7.6

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Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (41-43)

11. Sergeant, ESG. Ovine Johne’s disease in Australia – the first 20 years.

Aust Vet J. 2001;79:484-491.

12. De Lisle GW, Collins DM and Huchzermeyer HF. Characterization of ovine strains of Mycobacterium paratuberculosis by restriction endonuclease analysis and DNA hybridization. Onderstepoort J Vet Res.1992;59(2):163.

13. Shulaw WP, Bech-Nielsen S, Rings DM, Getzy DM and Woodruff TS. Serodiagnosis of paratuberculosis in sheep by use of agar gel immunodiffusion. Am J Vet Res 1993;54:13-19.

14. Cocito CP Gilot, Coene M, De Kesel M, Poupart P,Vannuffel P. Paratuberculosis. Clin Microbiol Rev 1994;7:328-345.

15. Collins MT. Diagnosis of paratuberculosis. Vet Clin North Am. Food Anim Pract. 1996;12:357-371.

16. Burnside DM, Rowley BO. Evaluation of an enzyme-linked immunosorbent assay for diagnosis of paratuberculosis in goats. Am J Vet Res. 1994; 55:465-466.

17. Dubash K, Shulaw WP, Bech-Nielsen S, Stills HF and Slemons RD. Evaluation of an agar gel immunodiffusion test kit for detection of antibodies to Mycobacterium paratuberculosis in sheep. J Am Vet Med Assoc. 1996;208:401-403.

18. Sweeney RW, Whitlock RH, Buckley CL, Spencer PA. Evaluation of a commercial enzyme-linked immunosorbent assay for the diagnosis of paratuberculosis in dairy cattle. J Vet Diagn Investig. 1995;7:488493.

19. Nielsen SS, Nielsen KK, Huda A, Condron R, Collins MT. Diagnostic techniques for paratuberculosis. In: Bulletin of the international dairy federation. IDF 2001;362:5–17.

20. Perez VJ, Tellechea JJ, Badiola MG, Garcia-Marin JF. Relation between serologic response and pathologic findings in sheep with naturally acquired paratuberculosis. Am J Vet Res 1997;58:799-803.

21. Sven B, John PB, Frank J, Griffin T. Autoreactive antibodies are present in sheep with Johne’s disease and cross-react with Mycobacterium avium subsp. paratuberculosis antigens. Microbes Infec. 2007;9:963-970.

22. Dubash K, Shulaw WP, Bech-Nielsen S, Stills HF, Slemons RD. Evaluation of an enzyme-linked immunosorbent assay licensed by the USDA for use in cattle for diagnosis of ovine paratuberculosis. J Vet Diagn Investig. 1995;7:347-351.

23. Hilbink F, West DM, DeLisle GW, Kittelberger R, Hosie BD, Hutton J, Cooke MM, Penrose M. Comparison of a complement fixation test, a gel diffusion test and two absorbed and unabsorbed ELISAs for the diagnosis of paratuberculosis in sheep. Vet Microbiol. 1994;41:107116.

24. Sockett DC, Conrad TA, Thomas CB and Collins M. T. Evaluation of four serological tests for bovine paratuberculosis. J Clin Microbiol.

1992;30:1134-1139.

25. Beth HN, Raúl GB. Mycobacterium avium subsp. paratuberculosis in Veterinary Medicine. Clin Microb Reviews 2001;14: 489-512.

26. Stewarta DJ, Vaughana JA, Stilesa PL, Noskea PJ, Tizarda MLV, Prowsea SJ, Michalskia WP, Butlerb KL and Jonesc SL. A long-term study in Merino sheep experimentally infected with Mycobacterium avium subsp. paratuberculosis: clinical disease, faecal culture and immunological studies. Vet Microb. 2004;104:165–178.

27. John PB, Valentina R, Stefania Z, Stefano R, Niyaz A and Leonardo AS. Antigenic profiles of recombinant proteins from Mycobacterium avium subsp. paratuberculosis in sheep with Johne’s disease. Vet Immunol Immunopatho 2008;122:116–125.

28. Darcel C and Logan-Handsaeme B. ELISA testing for antibody to Mycobacterium paratuberculosis. Can Vet J. 1998;39:335-336.

29. Sugden EA, Stilwell K and Michaelides AA. comparison of lipoarabinomannan with other antigens used in absorbed enzyme immunoassays for the serological detection of cattle infected with mycobacterium paratuberculosis. J Vet Diagn Investig. 1997;9:413417.

sufficient data to compare with the results of this study. The total positivity % (7.6%) is considered high as the disease now has a virtually worldwide distribution in farmed ruminants as well as many species of wild ruminants (1, 11), also the high S/P% for the all 7 positive sera indicate the high levels of antibodies against (Map) in the affected sheep (20, 22, 23), this mean that we need more investigations to confirm Johne's disease in sheep and also in other susceptible ruminants. In this study few numbers of male samples were included, only 3 samples were tested and all gives negative results 0.0%, because of the breading style in keeping few numbers of rams for each flock. This study has resulted in detection of antibodies against Mycobacterium avium subsp. paratuberculosis in sheep.

The study was done for the first time in Iraq to provide information about this disease for subsequent studies.

Acknowledgments This study was supported by College of Veterinary Medicine, University of Mosul. Mosul, Iraq.

References 1. Kennedy DJ and Benedictus G. Control of Mycobacterium avium subsp. paratuberculosis infection in agricultural species. Rev. sci.

Tech. Off. Int. Epiz 2001; 20, 151-179.

2. Navneet K D, Jeff E, Whittington RJ, Jenny-Ann LML. Risk factors for ovine Johne’s disease in infected sheep flocks in Aust. Preve Vet Med. 2007; 82:51–71.

3. Stehman SM. Paratuberculosis in small ruminants, deer, and South American camelides. Vet Clin North Am. Food Anim Pract. 1996;12, 441–455.

4. Reddacliff LA, McClure SJ and Whittington RJ Immunoperoxidase studies of cell mediated immune effector cell populations in early Mycobacterium avium subsp. paratuberculosis infection in sheep. Vet Immunol Immunopathol. 2004;97:149-162.

5. Sigurethardottir OG, Valheim M, PressCM. Establishment of Mycobacterium avium subsp. paratuberculosis infection in the intestine of ruminants. Adv. Drug Deliv. Rev. 2004;56:819-834.

6. Collins DM, Hilbink F, West DM, Investigation of Mycobacterium paratuberculosis in sheep by faecal culture, DNA characterization and the polymerase chain reaction. Vet Rec. 1993;133(24):599.

7. Satoko K, Deborah LT, Yasuyuki M, Whittington RJ. Detection of Mycobacterium avium subsp. paratuberculosis in ovine faeces by direct quantitative PCR has similar or greater sensitivity compared to radiometric culture. Vet Microb. 2007;125:36–48.

8. Clarke CJ, Patterson IA, Armstrong KE, Low JC. Comparison of the absorbed ELISA and agar gel immunodiffusion test with clinicopathological findings in ovine clinical paratuberculosis. Vet Rec.1996;139:618-621.

9. McKenna SL, Keefe GP, Barkema HW and Sockett DC., Evaluation of three ELISAs for Mycobacterium avium subsp. paratuberculosis using tissue and fecal culture as comparison standards. Vet. Microbiol 2005;110:105–111.

10. Quinn PJ, Carter ME, Markey B and Carter GR. Clinical Veterinary Microbiology. USA: Mosby, an imprint of Elsevier limited 2004;157.

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Effect of treating lactating rats with lead acetate and its interaction with vitamin E or C on neurobehavior, development and some biochemical parameters in their pups A. A. Hassan and H. M. Jassim Department of Physiology, Biochemistry and Pharmacology, College of Veterinary Medicine, University of Mosul, Iraq (Received February 17, 2009; Accepted October 5, 2009) Abstract The current study investigated the effect of administration of vitamin E (600mg/ kg diet) concomitantly with lead acetate (10mg/kg, orally) and vitamin C (100mg/kg, orally) concomitantly with lead acetate (10mg/kg, orally) to the female lactating rats on the neurobehavioral, landmarks development and some biochemical tests in their pups. Administration of lead acetate to the female lactating rats caused a significant increase in open field activity test including (the number of squares crossed and rearing test within 3 minutes), olfactory discrimination test, triglycerides and malondialdehyde brain tissue, with a significant decrease in glutathione brain tissue and high density lipoproteins in their pups. The present study demonstrated that treatment of female lactating rats with vitamin C and lead acetate produced a significant decrease in righting reflex test in their pups.

Administration of vitamin E concomitantly with lead acetate to the female lactating rats caused a significant increase in glutathione level accompanied with a significant decrease in malondialdehyde and triglycerides levels in their pups. The present study showed that treatment of female lactating rats with vitamin E or C with lead acetate produced a significant decrease in rearing test, whereas a significant increase in high density lipoproteins in their pups. It is concluded that administration vitamin E or C to the female lactating rats reverse the adverse effects produced by lead acetate on neurobehavioral. Vitamin E had positive effect on the levels of glutathione, malondialdehyde brain tissue, triglyceride and high density lipoproteins in their lactating pups.

Keywords: Vitamin E, Vitamin C, Lead acetate, Neurobehavior, Glutathione, Malondialdehyde, Rat pups.

Available online at http://www.vetmedmosul.org/ijvs

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The aim of present study is to assess the role of administration of vitamin E & C concomitantly with lead acetate to the dams during lactation period (21days) on the neurobehavioral, landmarks development and some biochemical changes in their pups.

Introduction Lead is an ubiquitous element in the environment, it is used in many industrial activities including mining, refining and producing lead â&#x20AC;&#x201C; acid batteries (1). Although this heavy metal is less widely used today, it remains a significant public health problem. Animals may be exposed to lead via contaminated food or water and fuel additives (2). The alimentary and respiratory tract are the major routes of lead entry into the body (3). Once the lead is in the bloodstream, it is distributed into soft and hard tissues (4). Milk is the most important food source for newborn, however, also be a pathway of maternal excretion of toxic elements such as lead, and these toxins impact most severely on the newborn at a time of rapid development of the central nervous system (5). In the nervous system, all neurons and glial cells form a very large network, integrate all external and internal stimuli and contributes to the elaboration of adequate responses (6). Lead enters the brain and selectively deposited in the hippocampus and cortex, as well as in non-neuronal elements that are important in the maintenance of the blood brain barrier function. Lead exposure causes distractibility inability to maintain physical balance and it affects some complex functions including learning (7). The neuropathlogical effects of lead include nervousness, anxiety and symptomatic encephalopathy at the end (8). Pervious study has shown that the level of lead in milk are thus similar to those in plasma (5). In rodents lead mobilized from the skeleton is transferred to the suckling offspring during lactation (9), and that lactational transfer after current or recent exposure to lead in dams was considerably higher than placental transfer (10). The main targets organ of lead toxicity are the red blood cell, central nervous system, peripheral nerves and the kidney (11).

Recent study reported that lead acetate can be transmitted through mother milk to their offspring. Lead acetate make bad effects on the reproductive systems of both males and females rats. While giving vitamin E as antioxidant found to have no improving effect in the treatment of lead acetate disturbances on the reproductive system of both sexes (12). Lead-induced oxidative stress contributes to the pathogenesis of lead poisoning for disrupting the delicate prooxidant/antioxidant balance that exists within mammalian cells. Production of reactive

Materials and methods Adult healthy albino rats were obtained from the animals house of the Veterinary Medical College â&#x20AC;&#x201C; University of Mosul at 3-4 months of age weighing 150200g. The rats were mated (3:1 females to male). Pregnant rats were removed and kept in separated polyproplyne cages under condition of temperature (22-26 C°) and lighting (12hours light /12hours dark). The rats were supplied a standard pellet diet and tap water ad libitum. At birth dams were separated with their pups from the first day of parturition.

In this experiment, animals divided into four groups: Group 1: dams (rats during lactation period) received normal saline orally as control group. Group2: dams received lead acetate at (10mg/kg B. Wt.) orally during lactation period, lead acetate dissolved in distalled water and given at 1 ml/kg (12). Group3: dams received lead acetate (10mg/kg B. Wt.) orally with concomitant administration of vitamin E at (600 mg /kg diet) during lactation period (14) (Shang Hang, China). Group 4: dams received lead acetate (10mg/kg B. Wt.) orally with simultaneous administration of vitamin C at (100 mg /kg B. Wt.) orally during lactation period (15) (Chemical Suppl, South Australia).

Twenty pups from each groups were selected randomly for examination. Pups were examined at the 7 day of age for neural behaviour which included righting reflex, cliff avoidance and olfactory discrimination tests (16). Central nervous system activity test were examined at the 21 day which include onset of movement test, open field activity test (17). This test measures the general locomotor activity, exploration which include (the number of squares crossed and rearing test within 3 minutes) and negative geotaxis test

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(16). Sensimotor system activity were examined which include approach response, touch response, click response and tail pinch response (18). Landmarks development tests included opening of ear, appearance of hair and teeth, opening of eyes, descending time of testis and appearance time of vaginal opening (16).

Blood was collected from pups at age 21 days for biochemical examination. Total cholesterol, high density lipoproteins, glucose, alanine aminotransfererase, aspartate aminotransfererase, and albumin, were measured using colorimetric assay kit (Syrbio, Syria) and triglyceride was measured using kit (bioMerieux, France), Pups weights monitored at birth and at weaning time. Pups were sacrificed at the end of experiment and the brain was placed in ice normal saline for glutathione (Moron method) (19) and malondialdehyde estimation (Gilbert method) (20).

Data were analyzed statistically using one way analysis of variance. Group differences were determined using Duncan multiple range test. Data of approach response, touch response, click response and tail pinch response analyzed statistically using Mann _ whitny _ U _ test.

Statistical significance was considered at (P < 0.05) (21).

acetate during lactation period did not effect significantly the negative geotaxis test in their pups compared with pups of lead acetate group. Table 1- showed that rats receiving lead acetate during lactation period cause a significant increase (P≤0.05) in rearing and the number of squares crossed tests within 3 minutes in their pups compared with the pups of the control group. Treatment with vitamin E & C to the rats receiving lead acetate during lactation period caused no significant changes in rearing test in their pups.

On the other hand, the number of square crossed test was significantly decrease by vitamin E & C in pups compared with the lead acetate group.

Table 2 shows that administration of lead acetate to the rats during lactation period caused a significant increase in olfactory discrimination (P≤0.05) in their pups compared with the pups of control group.

Treatment with vitamin E & C to the rats receiving lead acetate during lactation period did not affect significantly in olfactory discrimination test in their pups compared with the pups of lead acetate group. No significant changes between groups in cliff avoidance test. Same Table demonstrated that administration of lead acetate to the rats during lactation period did not effect significantly in righting reflex test in their pups compared with the pups of control values. Treatment with vitamin E of rats receiving lead acetate during lactation period did not affect significantly in righting reflex in their pups compared with the pups of lead acetate group, but treatment with vitamin C of rats receiving lead acetate during lactation period cause a significant decrease (P≤0.05) in righting reflex test in their pups compared with the pups of lead acetate group.

The data of vitamin E & C revealed no significant differences on sensimotor include (approach, touch, click and tail pinch responses),landmarks development and weighing of pups (at 21 days) from rats receiving lead acetate during lactation period are shown in Table 3,4,5.

Results In the current study Table 1- revealed that administration of lead acetate to the dams during lactation period did not effect the onset of movement and negative geotaxis tests in their pups compared the pups of control group. Treatment with vitamin E to the rats receiving lead acetate during lactation period did not effect significantly the onset of movement and negative geotaxis tests in their pups, but the treatment with vitamin C to the rats receiving lead acetate during lactation period produced a significant increase (P≤0.05) in the onset of movement test. Treatment with each of vitamin E & C to the rats receiving lead

Table 1. The onset of movement, open field activity tests and negative geotaxis in suckling pups at(21 days) from dams treated with lead acetate and their interaction with vitamin E or C for 21 consecutive days (lactation period).

Treatment of dams control Lead acetate (10mg/ kg) orally

Onset of movement/ sec ab 2.15±0.13 a 2.05±0.13 ab 2.30±0.14

Open field Rearing with 3 min square crossed with 3 mins a a 56.75±2.42 11.20±0.51 b b 65.25±3.48 14.50±0.48 b a 65.20±2.52 11.90±0.46

Lead acetate (10mg/ kg) orally + vitamin E (600mg/ kg diet) b ab Lead acetate (10mg/ kg) orally 2.55±0.18 58.35±1.96 + vitamin C (100mg/kg) Values were expressed as means ± SE from 20pups per treatment.

Values with different letters are significantly different at (P≤0.05).

47

a 12.05±0.65

Negative geotaxis/sec a 4.65±0.41 ab 5.80±0.51 b 6.7±0.64 ab 6.05±0.29

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Table 2. The righting reflex,cliff avoidance and olfactory discrimination tests in suckling pups at (7 days) from dams treated with lead acetate and their interaction with vitamin E or C for 21 consecutive days (lactation period).

Treatment of dams

Righting reflex(sec) b 2.95±0.13 b 2.90±0.17 ab 2.65±0.18

Control Lead acetate (10mg/ kg) orally

Lead acetate (10mg/ kg) orally + vitamin E(600mg/ kg diet) Lead acetate (10mg/ kg) orally a 2.30±0.11 +vitamin C(100mg/kg) Values were expressed as means ± SE from 20pups per treatment.

Values with different letters are significantly different at (P≤0.05).

Cliff avoidance (sec) a 9.35±0.57 a 8.75±0.89 a 10.25±0.95

Olfactory discrimination (sec) a 11.75±0.42 b 23.85±1.33 b 20.70±1.93

a 10.30±0.82

b 20.90±1.48

Table 3. The approach, touch, click, tail pinch responses in suckling pups at (21 days) from dams treated with lead acetate and their interaction with vitamin E or C for 21 consecutive days (lactation period).

Treatment of dams Control Lead acetate (10mg/ kg) orally

Approach response/ score a 1.90±0.02 a 1.90±0.02 a 1.90±0.02

Touch response/ score a 2±0 a 2.05±0.02 a 2.05±0.02

Click response/ score a 2.60±0.16 a 2.70±0.14 a 2.70±0.14

Tail pinch response/ score a 3±0.1 a 2.90±0.12 a 2.95±0.13

a 2±0

a 2.85±0.1

a 3±0.02

Lead acetate (10mg/ kg) orally + vitamin E (600mg/ kg diet) Lead acetate (10mg/ kg) orally a 1.95±0.02 + vitamin C (100mg/kg) Values were expressed as means ± SE from 20 pups per treatment.

Table 4. The landmarks development in suckling pups from dams treated with lead acetate and their interaction with vitamin E or C.

Treatment of dams Control Lead acetate (10mg/ kg)

Appearance of ear opening a 2.1±0.12 a 2.15±0.13 a 2.15±0.13

Appearan ce of hair

Appearan ce of teeth

Appearance of eye

Descending time of testis

a 6.4±0.21 a 6.7±0.16 a 6.4±.19

a 5.9±0.17 a 5.9±0.17 a 6±0.16

a 16.16±0.2 a 17.15±0.26 a 17.3±0.17

a 33.5±0.29 a 32.8±0.46

Lead acetate (10mg/ kg) + vitamin E (600mg/ kg diet) Lead acetate (10mg/ kg) a a a 2.1±0.12 6.6±0.

2 5.9±0.18 + vitamin C (100mg/kg) Values were expressed as means ± SE from 5 pups per treatment.

In the present study, Table 6 demonstrated that administration of lead acetate to the rats during lactation period cause a significant decrease in glutathione level with

a 17.10±0.2

a 33.15±0.37 a 33.25±0.33

Appearance of vaginal opening a 47.2±0.40 a 47.5±0.36 a 47.0±0.24 a 47.4±0.32

increase in malondialdehyde in brain tissue of their pups compared with the control group. Administration of vitamin E to the rats receiving lead acetate during lactation period

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produce a significant increased in glutathione and decrease (P≤0.05)in malondialdehyde levels in their pups compared with the pups of lead acetate group. No significant differences in the level of glutathione and malodialdehyde of the pups when their dams treated with vitamin C and lead acetate concomitantly during lactation period compared with the pups of lead acetate group. In same Table the data shows no significant differences in the levels of alanine aminotransferase, aspartate aminotransferase and albumin in the pups of all group.

Table 7 demonstrated that administration of lead acetate alone or concomitantly with vitamin E & C to the rats during lactation period did not effect significantly glucose and cholesterol levels in their pups. Administration of lead

acetate to rats during lactation period caused a significant increase (P≤0.05) in triglyceride level and significant decrease in high density lipoproteins in their pups compared with the pups of control value. Treatment with vitamin E to the rats receiving lead acetate during lactation period produced a significant decrease (P≤0.05) in triglyceride and significant increase in high density lipoproteins level in their pups compared with the pups of lead acetate group. Rats administered lead acetate with vitamin C during lactation period did not affect triglyceride level significantly in their pups compared with that of lead acetate group, on the other hand vitamin C caused a significant increase (P≤0.05) in high density lipoproteins in pups compared with the pups of lead acetate group.

Table 5. The body weight in suckling pups from dams treated with lead acetate and their interaction with vitamin E or C for 21 consecutive days (lactation period).

Treatment of dams

Weight(g) at 1 day a 5.46±0.02 a 5.44±0.02 a 5.33±0.10

Control Lead acetate (10mg/ kg) orally

Lead acetate (10mg/ kg) orally + vitamin E (600mg/ kg diet) Lead acetate (10mg/ kg) orally a 5.43±0.02 + vitamin C (100mg/kg) Values were expressed as means ± SE from 20 pups per treatment.

Weight (g)at 21 day a 25.27±0.19 a 25.31±0.45 a 25.06±0.15 a 25.13±0.16

Table 6. The glutathione, malondialdehyde brain tissue, alanine amimotraferase, aspartate amimotraferase and albumin in suckling pups from dams treated with lead acetate and their interaction with vitamin E or C for 21 consecutive days (lactation period).

Treatment of dams Control Lead acetate (10mg/ kg) orally

Glutathione µmlg

Malodialdehy de nm/g

a 2.73±0.2 c 1.19±0.17 b 1.8±0.15

b 107±4.19 a 181.8±8.39 b 112±6.71

Lead acetate (10mg/ kg) orally + vitamin E (600mg/ kg diet) Lead acetate (10mg/ kg) orally c a 0.99±0.002 167.4±9.9 + vitamin C (100mg/kg) Values were expressed as means ± SE from 5pups per treatment.

Values with different letters are significantly different at (P≤0.05).

Alanine Amimotraferase Unit/ L a 11.54±0.62 a 11.16±0.99 a 10.56±0.54

aspartate amimotraferase Unit/ L a 23.34±1.61 a 22.12±0.99 a 24.92±0.8

a 11.08±0.47

a 22.6±1.28

Albumin g/dl a 3.06±0.17 a 2.97±0.11 a 3.2±0.38 a 3.42±0.48

increase in the open field activity test including (rearing and the number of squares crossed tests within 3 minutes) and olfactory discrimination test in their pups compared to pups of the control group.

Discussion The present study showed that administration of lead acetate to rats during lactation period caused a significant

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The nervous system is the most sensitive target of lead exposure. Fetuses and young animals are especially vulnerable to the neurologic effects of lead because their brains and nervous system are still developing and blood brain barrier is incomplete (22). Lead neurotoxicity results in behavioral and neurochemical alteration in neurons as a result of changes and disruption of main structural components of the blood brain barrier, through primary injury to astrocytes and to secondary damage of the endothelial microvasculature (3). There are numerous studies utilizing experimental animal models on the central nervous system, These studies have mainly been concerned with possible effects of lead on certain performance tasks

that might reflects a cognitive function (learning and memory) or sensorimotor function in the infant animal exposed to lead very early in life or in utero (23). Some investigators studied effects of lead on the action of neurotransmitters using isolated peripheral nerve preparation, both cholinergic and adrenergic synaptic evoked transmitter released is inhibited by lead, and this effect is prevented by calcium (23). Lead affects primarily the metabolism of calcium (24), and inhibits the action of calcium as a result lead can affect calcium-dependent processes and interact with proteins including sulfhydryl, amine, phosphate, and carboxyl groups (21).

Table 7. The glucose, cholesterol, triglyceride and high density lipoproteins in suckling pups from dams treated with lead acetate and their interaction with vitamin E or C for 21 consecutive days (lactation period).

Treatment of dams Control Lead acetate (10mg/ kg) orally

glucose mg/dl a 102±6.5 a 106±3.7 a 104±4.3

Cholesterol mg/dl a 71.3±4.8 a 91.9±10.4 a 71.6±5.9

Lead acetate (10mg/ kg) orally + vitamin E (600mg/ kg diet) Lead acetate (10mg/ kg) orally a a 106±3 80.5±7.8 + vitamin C (100mg/kg) Values were expressed as means ± SE from 5pups per treatment.

Values with different letters are significantly different at (P≤0.05).

The nervous system is the most sensitive target of lead exposure. Fetuses and young animals are especially vulnerable to the neurologic effects of lead because their brains and nervous system are still developing and blood brain barrier is incomplete (22). Lead neurotoxicity results in behavioral and neurochemical alteration in neurons as a result of changes and disruption of main structural components of the blood brain barrier, through primary injury to astrocytes and to secondary damage of the endothelial microvasculature (3). There are numerous studies utilizing experimental animal models on the central nervous system, These studies have mainly been concerned with possible effects of lead on certain performance tasks that might reflects a cognitive function (learning and memory) or sensorimotor function in the infant animal exposed to lead very early in life or in utero (23). Some investigators studied effects of lead on the action of neurotransmitters using isolated peripheral nerve preparation, both cholinergic and adrenergic synaptic evoked transmitter released is inhibited by lead, and this effect is prevented by calcium (23). Lead affects primarily the metabolism of calcium (24), and inhibits the action of

Triglyceride mg/dl b 121±4.8 a 186.8±5.8 b 118.2±6.3

High density Lipoprotein mg/dl a 44.1±3.9 b 29.2±3.4 a 42.1±2.3

a 196±4.8

a 40.9±4.7

calcium as a result lead can affect calcium-dependent processes and interact with proteins including sulfhydryl, amine, phosphate, and carboxyl groups (21).

Neurotoxicity may be a consequence of alterations in cholinergic function mediated by the enzyme acetylcholinesterase (AchE) (25).

Maged recorded that lead caused a progressive decrease in the activity of acetylcholinesterase in different brain regions and spinal cord (26).

The enzyme inhibition is generally reached its significance after 10 to 20 days of lead acetate intake orally to the rabbits, such alteration in cholinergic transmission suggests that lead is able to reach the CNS and exerts its neurotoxic effect (26). It was supposed that oxidative stress was one possible mechanism for lead neurotoxicity. lead– induced oxidative stress might result from accumulation of 5-aminolevulinic acid (ALA), a potential endogenous source of free radical, induced by inhibition of lead to ALA dehydratase, overload of ALA seemed to be involved in the neurological disturbances, which leads to inhibition γaminobutyric acid (GABA) release from synaptosomes and blocking GABA receptor (27). Nihei et al (28) have

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production of lipid peroxide by scavenging free radicals in biological membrane (39).

Previous studies have revealed that vitamin E possesses an antioxidant activity in protecting cells from damage by highly reactive superoxide free radicals production (35). In the tissue of vitamin E deficient animals, it is reported that lipid peroxidation is enhanced suggesting that vitamin E plays a role as physiological antioxidant on its chemical properties, and prevent oxidation of low density lipoprotein (40). While vitamin C the most abundant water- soluble antioxidant in the body acts primarily in cellular fluid of particular in combating free radical for caused by pollution furthermore vitamin C help vitamin E to return to its active form (40). Moreover, Frei (41) found that only ascorbic acid is protectively enough to intercept oxidant in the aqueous phase before they can attack and cause detectable oxidative damage to lipids, as compared to many other lipophilic and hydrophilic antioxidants. (42) revealed that administration of vitamin E to rats during lactation period caused a positive effects on lipid profile, glutathione and malondialdehyde in brain tissue, of their offspring.

reported ALA can cause oxidative stress to rats brain.

Additionally, direct interaction of lead to biological membranes was to induce lipid peroxidation. Leadexposure might also induce decrease in activities of free radical scavenging enzymes. This mainly attributed to high affinity of lead to sulfhydryl-groups in these enzymes (29).

The result of the current study showed that administration of lead acetate to the rats during lactation period caused a significant increase in malondialdehyde and triglyceride with a significant decrease in glutathione in their pups compared with the control group. Numerous reports have documented increased lipid peroxidation (LPO) and decreased glutathione (GsH) and superoxide dismutase (SOD) activity in the brain homogenates of lead treated rats (30). Furthermore, lead exposure led to depletion of brain glutathione content, superoxide dismutase activity, and increase in thiobarbituric acid reactive substances (TBARS), and the activity of glutathione S-transferase bound enzyme (31). Oxidative damage associated with lead in the brain has been proposed as a possible mechanism of lead toxicity (31). Some investigators revealed that lead â&#x20AC;&#x201C;treated cultured aortic endothelial cells caused increase in the production of the lipid peroxidation products malondialdehyde and enhanced generation of hydroxyl radical compared with control cells which is considered as a direct cause of oxidative stress (33). It should be noted that hydroxyl radicals are primarily produced from sequential reduction of superoxide radical and hydrogen peroxide radical (33). Lead-induced oxidative stress associated with hyperglycemia suggested to contribute in the overproduction of very low density lipoprotein (VLDL), increasing the burden of triglyceriderich lipoproteins on the common lipolytic pathway at the level of lipoprotein in lipase (34).

In the current study administration vitamin E &C to the rats receiving lead acetate during the lactation period ameliorating the effect of lead acetate in their lactating pups. Recent study carried out effects of lead actate at 600 p.p.m. in drinking water during pregnancy and lactation caused significant decrease in activities of superoxide dismutase, glutathione peroxidaes and glutathione reductase in hypothalamus, corpora quadrigemina and corpus striatum in weaned pups (mouse) (27).

Vitamin E is necessary for the maintenance of normal neurological structures and function, and play a role in protecting lipid rich structures such as the brain from free radical (35). Antioxidant /chelating action represented by vitamin E improved the enzyme activity in the central nervous system (25). A major contributor to non-enzymatic protection of polyunsaturated fatty acid and low density lipoprotein from oxidation by free radicals against lipid peroxidation is vitamin E (35). Vitamin E as a lipid soluble, chain breaking antioxidant (37), plays a major protective role against oxidative stress (38), and prevents the

Acknowledgements This study was supported by the College of Veterinary Medicine, University of Mosul.

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26. Maged MY. Prophylactic efficacy of crushed Garlic lobes, Black seed Olive oils on cholinesterase activity in central nervous system parts and serum of lead intoxication rabbits. Turk J Biol. 2005 (29);173180.

27. Wang J. Junhui Wu. Zhang Z. Oxidative stress in mouse brain exposed to lead. Annals of Occupatinal Hygiene.2006;50:405-409.

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Use of saturated sodium chloride solution as a tissue fixative A. Al-Saraj Department of Dental Basic Sciences, College of Dentistry, University of Mosul, Mosul, Iraq E.mail : ayad-rehman@hotmail.com (Received March 4, 2009; Accepted October 5, 2009) Abstract The present study was carried out to examine the capability of saturated sodium chloride solution as a fixative agent instead of formalin which is regarded as a carcinogenic material. For this purpose 3 rabbits were used and their livers, kidneys and spleens were exposed and removed. Neutral buffered formalin solution, saturated sodium chloride solution and distilled water were used as fixatives for specimens obtained from the first, second and third rabbits respectively. Routine histological technique was performed to prepare a stained histological sections for light microscopic examination. The result showed that the tissue sections which were obtained by using sodium chloride have the same histological features and without any artifacts when they compared with the results obtained using formalin fixation method. We conclude that the saturated sodium chloride solution can be used as a fixative agent in some circumstances when no any fixative agent is available.

Keywords: Sodium chloride, Formalin, Tissue fixative.

Available online at http://www.vetmedmosul.org/ijvs

. $ # % &$# ' (%) # % * "#$ ! # , $ # 0, ,. / # ' % 0 / ,* % , 1 # . , , - , 6 ! 0, . * , ' 1 2 3 2 4 5 & # 6 4 (%)

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Introduction As early as 400 BC Hippocrates discussed the biological effects of mercury and alcohol as fixatives (1). The objective of fixation is to preserve cells and tissue constituents in as close a life-like state as possible and to allow them to undergo further preparative procedures without change. Fixation arrests autolysis and bacterial

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Iraqi Journal of Veterinary Sciences, Vol. 24, No. 1, 2010 (53-58)

A large variety of fixatives is now available but no single substance or known combination of substances has the ability to preserve and allow the demonstration of every tissue component. It is for this reason that some fixatives have only special and limited applications, and in other instances, a mixture of two or more reagents is necessary to employ the special properties of each. The selection of an appropriate fixative is based on considerations such as the structures and entities to be demonstrated and the effects of short-term and long-term storage (3). Each fixative has advantages and disadvantages, some are restrictive while others are multipurpose. Ferdinard Blum has been credited as the first person to use formaldehyde as a tissue fixative.

Formaldehyde, as 4% buffered formaldehyde (10% buffered formalin), is the most widely employed universal fixative particularly for routine paraffin embedded sections (4).

The aim of the present work is to test the possibility of using saturated sodium chloride solution as fixative and preserving agent during histological and pathological procedures and compared with the results obtained by using conventional formalin fixation.

Results Liver: fixed in neutral buffered formalin There were no tissue artifacts such as shrinkage, precipitate and swelling, therefore the liver appears to be divided clearly into many lobules, each one is hexagonal in shape with central vein located at its center. The portal canal which consist of a bile duct, a branch of hepatic artery and a tributary of the portal vein, all appeared clearly and enclosed in a common investment of connective tissue (take pink color). The paranchymal tissue of the liver appeared to be arranged in one or two cell thickness plates converging from the periphery to the center of lobule and the sinusoids are intermingled in between these plates. The cytoplasm appeared to be acidophilic and the nucleus basophilic and located at the center of the cells. Some hepatocytes appeared to contain more than one nucleus, and each nucleus contains a prominent nucleolus (Fig. 1 and 2).

Liver: fixed in saturated sodium chloride solution The sodium chloride solution was preserve the tissue and cells without any shrinking or swelling and without distorting or dissolving cellular constituents. Therefore the liver lobule architecture is similar to those of the control group. The central vein being located at the center of the hexagonal liver lobule. The hepatocytes appeared normal and arranged in form of plates. The cytoplasm appeared to be acidophilic while the nuclei basoplilic and some of hepatocytes were binucleated, this indicates that the tissue sections were taken the stained in good manner and without any precipitate (Fig. 3 and 4).

Materials and methods Three male rabbits were randomly assigned irrespective of age and weight into the following: First rabbit: specimens fixed in neutral buffered formalin (positive control rabbit). Second rabbit: specimens fixed in saturated sodium chloride solution. Third rabbit: specimens immersed in distilled water (negative control rabbit).

Each rabbit was anaesthetized with chloroform in an airtighted jar, then the animal was laid down on dissecting board. The liver, kidneys and spleen were exposed, removed and washed by water. Specimens of 5mm thickness of these organs were excised.

The fixation was made immediately after the removal of the above mentioned organs for 24 hours by using the following: First rabbit: Neutral buffered formalin fixative solution which composed of: Formaldehyde 37%, 100 ml. Distilled water 900 ml. Sodium phosphate monobasic (NaH2PO3), 4 gm.

Sodium phosphate bibasic (Na2HPO3), 6.5 gm.

Second rabbit: Saturated sodium chloride solution.

Third rabbit: Distilled water.

Liver: immersed in distilled water There was no section appear under microscope.

Kidney: fixed in neutral buffered formalin The cortex of the kidney showed clear cellular details of its all structures (renal corpuscles, convoluted and straight tubules) and stain well by (H &E) stain. There was no shrinkage or swelling of cells. While the medulla of the kidney also showed clear cellular details of it all structures (straight portions of tubules, thin segments of Henel`s loop and collecting tubules). There were no tissue artifacts (shrinkage, precipitate and swelling) and all of sections appeared to be stained well (Fig. 5 and 6).

Kidney: fixed in saturated sodium chloride solution Sections taken from kidneys of this rabbit revealed that the general structure of the kidney is nearly similar to that of the control group and seemed to be normal i.e. preserved cortex and medulla architecture without any morphological changes of the cells such as swelling and shrinkage or any artifacts. Also the cellular details appeared clearly and stained well (Fig. 7 and 8).

Then procedure of preparing the paraffin section slides and staining by Harrie`s Haematoxylin and Eosin (H&E) was performed to prepare a stained histological sections for light microscopic examination (5).

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Figure 1: Photomicrograph of the liver of rabbit fixed in neutral buffered formalin showing hepatocytes (H) and the sinusoids (S) (H&E X400).

Figure 4: Photomicrograph of the liver of rabbit fixed in sodium chloride solution showing the small bile duct (B) and the terminal branch of the portal vein (V) in the portal tract (H&E X400).

Figure 2: Photomicrograph of the liver of rabbit fixed in neutral buffered formalin showing the small bile duct (B), the terminal branch of the portal vein (V) and the terminal branch of hepatic artery (A) in the portal tract (H&E X400).

Figure 5: photomicrograph of the kidney (cortex) of rabbit fixed in neutral buffered formalin showing the glomerulus (G), proximal (P) and distal (D) convoluted tubules (H&E X400).

Figure 3: Photomicrograph of the liver of rabbit fixed in sodium chloride solution showing hepatocytes (H) and the sinusoids (S) (H&E X400).

Figure 6: photomicrograph of the kidney (medulla) of rabbit fixed in neutral buffered formalin showing the straight segment of the proximal tubule (SP) and the straight segment of the distal tubule (SD) (H&E X400).

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Figure 7: photomicrograph of the kidney (cortex) of rabbit fixed in sodium chloride solution showing the glomerulus (G), proximal (P) and distal (D) convoluted tubules (H&E X400).

Figure 10: photomicrograph of spleen of rabbit fixed in neutral buffered formalin showing the central artery (A) of the white pulp and the sinusoids (S) of the red pulp. (H&E X400).

Figure 8: photomicrograph of the kidney (medulla) of rabbit fixed in sodium chloride solution showing the straight segment of the proximal tubule (SP) and the straight segment of the distal (SD) tubule (H&E X400).

Figure 11: photomicrograph of spleen of rabbit fixed in sodium chloride solution showing the white (W) and the red (R) pulps (H&E X100).

Figure 9: photomicrograph of spleen of rabbit fixed in neutral buffered formalin showing the white (w) and the red (R) pulps (H&E X100).

Figure 12: photomicrograph of spleen of rabbit fixed in sodium chloride solution showing the central artery (A) of the white pulp and the sinusoids (S) of the red pulp. (H&E X400)

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However previous workers have indicated that exposure to formaldehyde has a serious effects and is highly suspected to be a human carcinogenic (9,10).

A method to overcome the problems of formaldehyde is to use an alternative fixative that is better suited for the preservation of tissues.

A study conclude that the cheap saturated table salt solution can be used as an alternative to the formaldehyde in preserving the bodies of animals (11).

Fixation of skin and lymph node fragments in anhydric sodium chloride at room temperature for periods of weeks or months was found to preserve morphological structures and immunoreactivity (12).

Segments of rat's aorta were harvested, immersed in pulverized dehydrated sodium chloride, and stored for 1 week to 3 months. Thereafter, they were desalinated and transplanted. The tensile strength and maximum intraluminal pressures did not significantly differ from freshly harvested, transplanted aortic segments (13).

Human skin fragments can be preserved in anhydric sodium chloride at room temperature for periods of weeks or months and successfully transplanted, retaining normal morphological structure (14).

The result of the present study showed that the saturated sodium chloride solution can be used as a fixative for tissue in pathological or histological procurers. The tissue sections which obtained have the same histological features when they compared with the results obtained using conventional formaldehyde fixation methods. It penetrates tissue but is relatively slow and prevents autolysis as well as it cause no precipitation of pigment in the tissues nor morphological changes (swelling or shrinkage) of the cells.

The exact mechanism by which the sodium chloride act on the tissue is unclear and no one mentioned its precise action on the tissue. Therefore it need more investigations and can be classified as unknown mechanism fixative, such as mercuric chloride, picric acid.

We suggest that this new (cheap and safe) method of fixation can be considered as an alternative fixative procedure which can be used in emergency cases when no any fixative is available (for pathologist, veterinarian, biologistâ&#x20AC;Śetc) in such cases for tissue preservation.

Kidney: immersed in distilled water There was no section appear under microscope Spleen: fixed in neutral buffered formalin Histological section throughout the spleen showed a well defined cellular structures arranged into white (splenic nodules and central artery) and red (splenic cords and sinuses) pulps without any artifacts (Fig. 9 and 10).

Spleen: fixed in saturated sodium chloride solution Microscopically, there was a preserved normal histological sections architecture nearly similar to those of control group without any morphological (shrinkage or swelling) changes. The well defined histological structures indicates well stained and preserved sections (Fig. 11 and 12).

Spleen: immersed in distilled water There was no section appear under microscope.

Discussion A systematic study of the fixatives began in the latter half of the 19th century, however, it must be noted that fixation by itself introduces a major artifact. Much attention was focused on developing fixatives that would preserve cells and tissue constituents in as close a life-like state as possible while allowing them to undergo further preparative procedures without change (6).

The mechanisms by which fixatives act may be broadly categorized as dehydrants, heat effects, cross-linkers, and effects of acids and combinations of these. Agents that combine with proteins are called additives and those that precipitate proteins are called coagulants. At this time, it is accepted that no one fixative fulfils all of the aims of cell or tissue preservation: namely prevention of autolysis and preservation of physical and chemical properties of the tissue (7).

In aldehydes include formaldehyde (formalin) the tissue is fixed by cross-linkages formed in the proteins, particularly between lysine residues. This cross-linkage does not harm the structure of proteins greatly, so that antigenicity is not lost. Formalin penetrates tissue well, but is relatively slow and the standard solution is 10% neutral buffered formalin. A buffer prevents acidity that would promote autolysis and cause precipitation of formol-heme pigment in the tissues (8).

Formalin is used for all routine surgical pathology and autopsy tissues when (H and E) slide is to be produced. It is the most forgiving of all fixatives when conditions are not ideal, and there is no tissue that it will harm significantly.

Most clinicians and nurses can understand what formalin is and it smells bad enough that they are careful handling it.

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10. AL-Saraj A, AL-Hubaity A. Histological and histopathological changes of lung of rats during different periods of formaldehyde exposure. Iraqi J Vet Sci.2003;17(2):111-21.

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5. Anderson G, Gordon K. Theory and Practice of Histological Techniques. 4th ed. Churchill Livingstone, Edinburgh,1999, pp: 47-67.

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