Diagnosis of major poultry diseases by Grupo Asís

PRESENTATION

BROCHURE

Diagnosis of Major Poultry Diseases Y. M. SAIF H. TORO

Diagnosis of Major Poultry Diseases

Diagnosis of Major Poultry Diseases Y. M. SAIF H. TORO

AUTHORS: Yehia “Mo” Saif, Haroldo Toro. FORMAT: 22 x 28 cm. NUMBER OF PAGES: 112. NUMBER OF IMAGES: 150. BINDING: hardcover.

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Handy and rigorous book focused on the diagnosis of the main diseases affecting poultry through a practical and visual approach of the topic, and written by prestigious experts with a wide experience in this field. Each disease has been thoroughly reviewed including the most updated information (clinical signs, gross and microscopic pathology, as well as methods of isolation or detection of the etiologic agent(s) and serologic methods for antibody detection) to better understand the content. Numerous graphic resources (images, graphs, tables, flowcharts) have been included to complement the information provided and make the contents understandable and accessible to readers.

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Diagnosis of Major Poultry Diseases

Presentation of the book This book is intended to be a concise reference book for the benefit of the practicing veterinarian and the laboratory diagnostician. It provides concise description of the major diseases affecting poultry. Both traditional and state-of-the-art molecular techniques used for diagnosis are described. Prevention, control and treatment of diseases are dependent on an accurate diagnosis. Hence, it is important that an accurate diagnosis is secured promptly. Delay in securing an accurate diagnosis could have serious consequences. A variety of complementary methods are usually used to reach a diagnosis. That would include field approaches such as clinical observations and gross lesions and laboratory investigations including microscopic lesions, isolation, detection of etiologic agent(s), antigens, or nucleic acid. Other diagnostic techniques include serologic testing for antibodies to suspected etiologic agents. In chapter 1, we provide the basis and rationale for these tests and their pros and cons and interpretation of results. Four groups of diseases (viral, bacterial, fungal, and protozoal) are presented in the following 25 chapters. These chapters have a standard format of a brief introduction including the etiology of the disease followed by the main emphasis of the chapter, which is the diagnosis of the disease. For each disease, the clinical signs, gross and microscopic pathology are described. Methods of isolation or detection of the etiologic agent(s) are described followed by the applicable serologic methods for antibody detection. The authors

The authors Yehia “Mo” Saif Dr. Yehia “Mo” Saif is Professor and Head Emeritus of the Food Animal Health Research Program of the Ohio Agricultural Research and Development Center (OARDC) at The Ohio State University (OSU). Dr. Saif received his DVM degree from Cairo University and a PhD from OSU. Among his research accomplishments were the first demonstration of the interspecies transmission of influenza viruses and coronaviruses, elucidating the molecular basis of interspecies transmission of influenza viruses, first demonstration of the diversity of antigenic types of infectious bursal disease viruses, discovery of several avian enteric viruses and definition of their pathogenesis and epidemiology, providing first description of the production and metabolism of the three turkey immunoglobulins elucidating the pathogenesis and epidemiology of Mycoplasma meleagridis including its venereal route of transmission, establishment of a specific-pathogen-free turkey flock in 1964 that has been in existence since and used for experimental studies, demonstrating the effect of relative humidity on the pathogenesis of aspergillosis. Dr. Saif is a past president of the American Association of Avian Pathologists (AAAP) and a Diplomate and past Chair of the Board of Governors of the American College of Veterinary Microbiologists (ACVM) and the American College of Poultry Veterinarians (ACPV). He is currently Editor of Avian Diseases and was Editor-in-Chief of the textbook Diseases of Poultry. He is a highly regarded expert on avian pathology and an advisor to the World Animal Health Organization (OIE), and his laboratory is an official OIE reference laboratory. He is author or coauthor of 170 peer-reviewed scientific articles, 28 book chapters and 264 abstracts and popular articles, and he mentored 31 graduate students. Dr. Saif has received numerous awards including AAAP Life Member and Special Service Award, the ACVM Distinguished Veterinary Microbiologist Award, an honorary diploma from the American Veterinary Epidemiology Society, and was elected an inaugural member of the Hall of Honor of the World Veterinary Poultry Association (WVPA) and the research excellence awards from OSU College of Veterinary Medicine (CVM), OARDC, AAAP, American Veterinary Medical Association (AVMA), and graduate teaching excellence from CVM.

Diagnosis of Major Poultry Diseases

Haroldo Toro

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Dr. Haroldo Toro is Professor in the Department of Pathobiology at Auburn University. He received his DVM degree in 1983 from the University of Chileâ&#x20AC;&#x2122;s College of Veterinary Sciences. In 1987, Dr. Toro received his PhD from the Institute of Avian Diseases of the Justus-Liebig-University in Giessen, Germany. Dr. Toroâ&#x20AC;&#x2122;s current research focuses on further understanding chicken anemia virus and its interactions with other avian pathogens, and further understanding the role of infectious bronchitis virus in respiratory disease of commercial chickens. During his career, Dr. Toro has also focused on other relevant avian diseases including for example avian influenza and inclusion body hepatitis. He has published 93 articles in peer-reviewed international scientific journals and is a member of the editorial board of the Avian Diseases journal. Dr. Toro was awarded the Pfizer Award for Research Excellence in 2007, the L. G. Wolfe Award for Excellence in Graduate Education in 2013, the Auburn University Alumni Professorship in 2013, and the P. P. Levine Award by the American Association of Avian Pathologists (AAAP).

Communication services Website Online visualisation of the sample chapter. Presentation brochure in PDF format. AuthorÂ´s CV. Sample chapter compatible with iPad.

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Diagnosis of Major Poultry Diseases Y. M. SAIF H. TORO

Table of contents 1. Principles of diagnosis in poultry (M. J. Pantin-Jackwood)

2. Viral diseases Newcastle disease and other paramyxovirus infections (E. Spackman) Infectious bronchitis (V. L. van Santen and H. Toro) Infectious laryngotracheitis (G. Beltrán and M. García) Avian influenza (L. Wang and Y. Zhang) Infectious bursal disease (Y. M. Saif) Marek’s disease (J. Dunn) Avian leukosis (G. Zavala) Chicken infectious anemia (H. Toro) Reovirus infections (E. Spackman) Metapneumovirus infections (Q. Yu) Fowlpox (D. N. Tripathy) Adenoviral diseases (F. W. Pierson) Enteric viral infections (Y. M. Saif) Avian encephalomyelitis (H. L. Shivaprasad)

3. Bacterial diseases Salmonellosis (R. Gast and W. D. Waltman) Fowl cholera (M. Prarat and J. Cui) Mycoplasma infections (M. El-Gazzar) Infectious coryza (C. L. Hofacre, M. Lee, J. R. Glisson and G. Zavala) Bordetellosis (K. B. Register) Ornithobacterial infections (H. M. Hafez) Clostridial diseases (K. V. Nagaraja and A. Thachil) Campylobacteriosis (I. Kassem and G. Rajashekara)

4. Fungal diseases Aspergillosis (M. Franรงa and H. L. Shivaprasad)

5. Protozoal diseases Coccidiosis (R. Hauck) Histomoniasis (R. Hauck)

VIRAL DISEASES

Newcastle disease and other paramyxovirus infections E. Spackman

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Newcastle disease and other paramyxovirus infections

Newcastle disease virus (NDV) is a form of avian paramyxovirus type 1 (APMV-1) that is highly virulent for chickens and turkeys. There are currently 13 recognized serotypes of avian paramyxovirus, but APMV-1, including NDV, is the most important for poultry. Newcastle disease (ND) is considered to be one of the most economically important diseases of poultry production worldwide, and is reportable to the World Organisation for Animal Health (OIE). NDV is endemic in many areas of Asia, South America, Africa and the Middle East, and can be carried asymptomatically by wild waterfowl. Numerous avian species are susceptible to infection with APMV-1. There are variations among APMV-1 strains in terms of virulence, and strains are classified as lentogenic, mesogenic, or velogenic. Lentogenic strains cause respiratory disease, but the most concern is with velogenic NDV, which cause severe disease.

Mesogenic NDV strains are “between” velogenic and lentogenic forms regarding severity of disease caused. In chickens, disease caused by velogenic NDV is very severe. Compared to chickens, turkeys are somewhat less susceptible to disease caused by some APMV-1 strains. There is little data on APMV-1 infection in game birds, but most species appear to be susceptible to infection and disease. APMV-1 rarely causes disease in waterfowl. Pigeons may be infected with pigeon-adapted strains of APMV-1, which tend to be mesogenic in poultry. In all species for which there is data, age seems to be of primary importance in the clinical severity of disease; younger birds experience the most severe disease. As with any disease, environmental stress factors and exposure to other infectious agents can also have a substantial effect on clinical outcome. There is no treatment for NDV, but good sanitation and biosecurity measures can reduce the risk of exposure. Vaccination, often with live non-virulent APMV-1 strains, is virtually universal in commercial chicken and turkey production. However, vaccines are not readily available for smaller

E. Spackman flocks, which remain unvaccinated. Good quality APMV-1 vaccines protect against disease and mortality from NDV when applied properly. In addition to NDV and less virulent APMV-1 strains, APMV type 2 (APMV-2), APMV type 3 (APMV-3), and APMV type 7 (APMV-7), although relatively rare, can cause disease in turkeys and chickens.

Diagnosis Clinical signs Most information on ND is from chickens and turkeys, but clinical presentation seems to be similar among avian species. Birds can become infected and clinically ill at any age, although disease tends to be more severe in younger birds. Clinical disease generally begins 2-3 days post-infection. Disease presentation varies among NDV strains. Some strains tend to cause visceral lesions (Fig. 1), while others cause neurological signs. Thus, NDV can be classified into viscerotropic and neurotropic pathotypes. Reductions in feed and water intake may be observed early after infection with either pathotype. Drops in egg production occur in breeders and layers.

Figure 1. Chicken with viscerotropic ND 3 days after exposure.

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(Fig. 2). Birds may still eat and drink if the neurological impairment does not prevent access to feed and water. Morbidity will be high, but mortality may not reach 100 % if birds can eat and drink. Mean death times are typically longer with neurotropic strains than with viscerotropic strains. The clinical signs of NDV infection can be very similar to those of highly pathogenic avian influenza (HPAI) virus infection and the rapid mortality can also resemble acute toxemia. Therefore, conclusive diagnosis can only be made with confirmation by laboratory tests. Figure 2. Chicken with torticollis caused by ND. Torticollis is relatively common, but not pathognomonic for ND. Image courtesy of Dr. K. Dimitrov.

Viscerotropic strains induce severe lethargy and, as the infection progresses, the birds become more listless and reluctant to move. Mortality can approach 100 %. Other common signs include ruffled feathers, drooping wings, mucus in the mouth and nasal cavity, respiratory distress from the mucus, green diarrhea, and edema of the head, comb and wattles. The comb and wattles may become cyanotic and necrotic. Disease is systemic and mortality can occur very suddenly, without apparent disease. The length of time between infection and death is typically 4-5 days. Infection with neurotropic strains results in clinical signs characterized by torticollis, tremors, ataxia or paralysis

Disease caused by APMV-2 and APMV-3 is less well characterized, but respiratory disease of varying severity and decreased egg production resulting from natural and experimental infections of chickens and turkeys have been reported.

Gross lesions The presence and type of gross lesions correspond with clinical disease and the virus strain causing the infection. Chickens infected with viscerotropic strains present with hemorrhages and necrosis in intestines (commonly at the cecal tonsils), spleen, thymus and proventriculus (often near the junction with the gizzard) (Fig. 3). The spleen may appear enlarged and mottled. Edematous ovaries, yolk peritonitis and hemorrhages in the reproductive tract of layers may be observed. Petechial hemorrhages in the trachea have been reported, but are uncommon. The air sacs may be thickened or sudsy if secondary bacterial infection is present. In contrast, gross lesions are often completely absent following infection with neurotropic strains, including a lack of lesions in central nervous system (CNS) tissues.

Histopathology Tissue collection for microscopic evaluation should include tissues with gross lesions (e.g. hemorrhages) or which correspond to the clinical manifestation (e.g. CNS tissue when neurological signs are evident).

Figure 3. Hemorrhagic proventriculus in a NDV-infected chicken. In severe cases, ulcers may be seen near the junction with the esophagus. Image courtesy of Dr. P. Miller.

However, because of the non-specific nature of NDV-induced microscopic lesions, histopathological evaluation has limited diagnostic utility.

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NEWcaSTLE DiSEaSE aND oTHEr ParaMYxoViruS iNFEcTioNS

Figure 4. Pharynx from a 4-week-old chicken infected with NDV, showing ulceration and erosion of the epithelium (hematoxylin and eosin stain, 10x magnification). Image courtesy of Dr. L. Susta.

Viscerotropic strains cause necrosis, ulceration and hemorrhage in a variety of organs including intestine, pancreas, spleen, and occasionally liver and gallbladder (Fig.Â 4). Hemorrhages are typically located in intestinal lymphoid patches. Lymphoid depletion has also been observed in the thymus, bursa, spleen and lymphoid patches in other organs (Fig.Â 5). Myocarditis has been reported rarely. Lesions are generally not seen in the lung. Histopathological lesions due to infection with neurotropic strains are usually observed only in the CNS. Lesions include neuronal degeneration, perivascular cuffing and gliosis.

Virus isolation and detection Virus isolation and diagnostic testing for APMV are typically conducted with oral and/or cloacal swabs, which are the preferred sample type regardless of detection test. Oral swab collection should include swabbing the entire oral cavity, including the choanal cleft. Swabs should be placed into a viral transport medium, preferably one containing protein (e.g. brain heart infusion broth). It is also critical to maintain a cold chain when transporting samples to the laboratory. If tissue testing is desired, organs where gross lesions are present or CNS tissue from birds with neurological signs should be collected.

Figure 5. Cecal tonsils from 4-week-old chickens. (a) Non-infected chicken; (b) Chicken infected with NDV, showing multifocal necrosis and lymphoid depletion (hematoxylin and eosin stain, 10x magnification). Images courtesy of Dr. L. Susta.

In most cases, samples will initially be screened for NDV by real-time reverse transcription polymerase chain reaction (RT-PCR) followed by virus isolation on positive samples. Numerous real-time and conventional RT-PCR tests are available and can identify specific strains. No single test can identify all APMV-1 strains and separate tests must be run for APMV-2 and APMV-3, so multiple tests may be conducted with a single set of samples. In some cases, virus isolation may be attempted directly. Once a suspected NDV-positive sample is identified, gene sequencing is

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used to help identify the pathotype and origin of the virus strain. Because NDV is an OIE-reportable disease, in vivo pathotyping of suspect cases will often be conducted by regional reference (OIE) laboratories.

Serology Antibodies to APMV-1 can be detected by hemagglutination inhibition assay (HI) or commercial enzyme-linked immunosorbent assay (ELISA). Several commercial ELISA kits are available. However, serology has limited application for diagnosis for several reasons. First, the virus will often kill birds before measurable antibodies can develop. Besides, the tests cannot distinguish antibodies resulting in response to NDV infection from those present due to vaccination or prior natural exposure to a non-pathogenic APMV-1 strain. Commercial tests are not currently available for APMV-2 and APVM-3 antibody detection. It should be noted that APMV-3 antibodies can cross-react with APMV-1 on some serological tests.

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2 Infectious bronchitis (IB) is a worldwide economically important disease of commercial chickens caused by a coronavirus. It is predominantly a respiratory disease, but can also affect oviduct and kidneys. Economic losses in broilers result from poor performance, increased mortality, and increased condemnations at the processing plant. Economic losses in breeders and layers result from drops in egg production, and reduced egg quality and hatchability. IB is controlled in breeders and layers by vaccination with type-specific live attenuated vaccines followed by boosting with inactivated virus, and in broilers by vaccination with live attenuated vaccines. Live attenuated vaccines are usually administered by spray or drinking water. Outbreaks of disease continue to occur despite vaccination due to the distinct ability of IB virus (IBV) to evolve and circumvent extensive vaccination programs with typespecific vaccines.

Diagnosis Diagnosis of IB is based on clinical signs, gross and microscopic lesions, virus isolation, detection of IBV RNA, detection of IBV antigen, and serological monitoring. Identification of the serotype of circulating strains is distinctively important in order to select the most appropriate type-specific vaccines to control the disease.

Clinical signs and gross lesions IBV clinical signs and lesions are not pathognomonic and vary depending on the virus strain, and age and type of chickens. Morbidity is typically near 100 % and chickens of all ages exhibit respiratory signs of varying severity characterized by gasping, coughing, sneezing, tracheal rales, and nasal discharge. Young chickens show depression, reluctance to eat and drink, huddling near heat sources, and increased mortality.

Infectious bronchitis V. L. van Santen and H. Toro Additional signs of infection with nephropathogenic IBV strains include wet droppings, increased water consumption, and varying mortality rates, which may reach 90 %. Layer and breeder hens may exhibit respiratory signs so mild that they are overlooked, but show a decline in egg production and quality (misshapen eggs, poor egg shell quality, and altered albumin consistency). Cold stress, mycoplasma infections and/or secondary bacterial infections increase the severity of respiratory signs and mortality rates in all ages and types of chickens. Differential diagnosis of IBV respiratory signs includes mesogenic Newcastle disease (ND), low pathogenic avian influenza (LPAI), mild forms of infectious laryngotracheitis (ILT), infectious coryza (IC), and mycoplasmosis. Differential diagnosis of decline in egg production includes mesogenic ND, adenovirus-caused egg drop syndrome (EDS) (in regions where present) and picornavirus (avian encephalomyelitis [AE]) infection in laying hens. Gross pathology in the upper respiratory tract includes exudate in the trachea, sinuses, and nasal passages, and foamy or cloudy air sacs (Fig. 1). Nephropathogenic strains cause swollen and pale kidneys, with tubules and ureters distended with uric acid. Differential diagnosis includes salt intoxication, severe dehydration, and other pathogens causing nephritis.

Histopathology Histological lesions in the trachea include deciliation, epithelial cell necrosis, massive infiltration of lymphocytes into the lamina propria, and hyperplasia of epithelial and Goblet cells (Fig. 2). Microscopic lesions in the kidney are those characteristic of interstitial nephritis.

Virus isolation IBV isolation is currently not routinely conducted for diagnostic purposes. If needed, IBV can be isolated from tracheal or choanal swabs, or tissue samples (trachea, kidney, oviduct), which must be refrigerated immediately and during transport to the laboratory for successful virus

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Chicken infectious anemia

Chicken infectious anemia determines economic losses to the poultry industry from increased mortality, reduced performance during rearing, increased culling, lower uniformity, and increased condemnations at slaughter. In addition, chicken anemia virus (CAV) causes transient immunodeficiency associated with increased susceptibility to and severity of health problems commonly absent in immunocompetent flocks. CAV transmission is both vertical and horizontal. Eradication is currently not possible because the virus is extremely resistant to both physical and chemical agents. Thus, challenge from ubiquitous CAV is virtually certain in poultry environments. The control of CAV in commercial broilers and layers is directed at generating high and long-lasting specific antibody responses in breeder flocks with the goals of impeding CAV vertical transmission and transferring specific maternal antibody to protect progeny chickens. Generation of immunity in breeders is achieved by vaccination. Some operations rely on natural infection based on the knowledge that all chicken premises are most certainly contaminated with CAV. This theoretically sound concept, however, often produces varying or suboptimal results in the industry. Breeder flocks with uneven immunity result in a few vertically-infected progeny chickens, which in turn will horizontally infect birds with less-than-optimal antibody levels, and thus reduce the average flock performance.

Diagnosis Diagnosis of chicken anemia is based on clinical signs, gross lesions, histopathology, viral DNA detection by polymerase chain reaction (PCR), virus isolation, and serologic monitoring. Signs and lesions vary considerably, depending on age (4-week-old or older chickens generally do not show clinical signs), viral dose, immune status of the host, and other individual factors.

H. Toro

Clinical signs Clinical signs induced by CAV infection include reduced weight gain and anemia. CAV intramuscular inoculation in 1-day-old chickens, an inoculation route that likely resembles vertical transmission, results in reduced weight gain usually noticeable around 10 days post-inoculation. In contrast, reduced weight gain is seen around 18 days after oral inoculation. Reduced weight gain results in uneven commercial broiler flocks with varying percentages of small birds. A portion of these small birds is selected for culling before harvest and another portion is subjected to condemnation at the slaughter plant. A similar timeline is observed for anemia; i.e., a noticeable reduction of hematocrit begins around day 10 after intramuscular inoculation, while orally-inoculated chickens show this reduction around day 18 post-inoculation. Normal hematocrit values in chickens vary significantly (30 % to 50 %) depending on different factors including genetic line, gender, body weight, and age. Young chickens infected with CAV show hematocrits of <25 % but, in severe cases, hematocrits of individual birds can reach values as low as 15 %. Thus, hematocrit values have been used frequently for diagnostic purposes both in experimental and commercial settings.

Gross lesions The most typical and characteristic gross lesions observed in CAV-infected chickens include atrophy of the thymus and pale bone marrow (Fig. 1). Other gross lesions include petechial and/or ecchymotic hemorrhages in muscles and other tissues. Atrophy of the thymus is bilateral and can be easily diagnosed by comparison with the thymus of healthy animals. In severe cases, the thymus almost disappears to the naked eye. The presence of pale bone marrow is a consequence of CAV causing destruction of hemocytoblasts. Inspection of bone marrow can be

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ChiCken infeCtious anemia

Figure 1. Chicken anemia gross lesions. (a) Healthy thymus. (b) Thymus atrophy in a chicken infected with CAV.

easily performed in commercial chickens by simply cutting through the femur or other bones containing bone marrow. Instead of the characteristic red color of normal bone marrow, CAV-affected chickens show yellowish discoloration and/or paleness. Ecchymotic and petechial hemorrhages seen in thighs, breasts, and other tissues of CAV-infected chickens are the result of alteration of blood clotting due to reduced platelets from thrombocytopenia.

decrease in cortical lymphocyte density or relative thickness of the cortex to the medulla) (Fig. 2c). A more objective method to evaluate lymphocyte depletion in the thymus uses histomorphometry. Morphometry is conducted on a black and white digital image of a section of a representative thymus lobule using ImageJ software (http://rsb. info.nih.gov/ij/).

Histopathology

Virus isolation and CAV DNA detection by PCR can be conducted from most tissues of CAV-infected chickens, but is most commonly conducted from thymus or liver samples. Virus isolation is labor-intensive, requires distinct cell cultures for successful virus replication, and is not performed routinely for CAV diagnostic purposes. Successful virus replication in cell cultures can be confirmed by immunofluorescence or immunohistochemistry (IHC) with reference antisera, or detection of CAV genomes by PCR. Virus isolation can also be performed by inoculation of specific pathogen-free (SPF) chickens free of CAV antibodies.

Histopathological changes are most easily observed in the thymus, as CAV causes different levels of lymphocytoblast depletion in the thymic cortex (Fig. 2). In addition, infected thymocytes show intranuclear inclusion (INI) bodies and apoptosis. Assessment of lymphocyte depletion is usually performed by histologic scoring.

Thymus lymphocyte depletion in formalin-fixed hematoxylin and eosin-stained thymus sections is scored as follows: score 1 = histologically normal (Fig. 2a); score 2 = mild depletion (5-20 % decrease in cortical lymphocyte density or relative thickness to the medulla) (Fig. 2b); score 3 = moderate to marked depletion (25-75 % decrease in cortical lymphocyte density or relative thickness of the cortex to the medulla); score 4 = severe depletion (>75 %

Virus isolation and detection

Different variations of PCR as well as quantitative PCR (qPCR) (or real-time PCR) are effectively used to detect CAV DNA in infected chicken tissues, fluids, and FTA® cards. These methods are highly sensitive and of great diagnostic value when supplemented with characteristic clinical and pathological findings. However, CAV has been shown to persist in infected birds. Thus, the sole detection

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of CAV DNA is insufficient to attribute clinical signs to CAV in commercial chickens. In contrast, PCR-associated techniques may prove essential for monitoring SPF chicken flocks intended to maintain a CAV-free status.

Serology Although CAV causes immunodeficiency, infected birds effectively generate CAV-specific antibody responses. Seroconversion in infected chickens becomes detectable 2 weeks after intramuscular inoculation. Antibody responses after oral inoculation require longer, presumably because the virus has to overcome additional barriers before successful establishment in the host. b

Figure 2. Lymphocyte depletion in the thymic cortex in chickens infected with CAV. (a) Healthy thymus with lymphocyte-rich (dark stained) cortex area and a lighter medulla area. (b) Mild- and (c) severe-lymphocyte depletion in the thymic cortex. Images courtesy of Dr. K. Joiner.

Regular monitoring of breeder flocks for presence and homogeneity of CAV antibody levels is important to assess vaccination success and assure homogeneous protective maternal antibody levels transferred to progeny chickens. Continuous antibody monitoring in breeders is also relevant, as older breeders (50-55 weeks of age) tend to produce increased percentages of progeny chickens negative for CAV antibodies. CAV antibody monitoring can be performed by different serological procedures but the enzyme-linked immunosorbent assay (ELISA) has been adopted worldwide by the poultry industry as the most efficient and cost-effective monitoring tool.

A minimum of 20 serum samples is recommended for broiler flocks and 30 for breeder flocks for adequate representation of the serologic status. The correct serum dilution to be used in ELISA is indicated by the instructions provided by the companies producing the ELISA kits. Use of serum dilutions lower than recommended may increase the number of false-positive results and lead to overestimation of vaccination success, while high serum dilutions may increase false-negative results and lead to the opposite conclusion. Companies producing CAV ELISA kits also provide software that allows classification of animals based on antibody titer ranges. These titer ranges vary from values considered negative to levels considered protective. Assessment of homogeneity can be performed directly from the values displayed in the software or by further statistical analyses including e.g. the coefficient of variation.

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