Immunosuppressive diseases of poultry

The book features an extensive collection of color photographs depicting the gross and microscopic images and the text provides background information and practical tips in identifying immunosuppression in poultry under laboratory and field conditions.

IMMUNOSUPPRESSIVE DISEASES OF POULTRY

This book is intended for diagnosticians and poultry veterinarians to aid in the identification and control of immunosuppressive diseases. The goal of this work is to present the reader with the most common diseases that can produce immunosuppression in poultry, provide guidance for the diagnosis of immunosuppressive diseases, and examine the challenges that a diagnostician may face in confirming a diagnosis of immunosuppression.

It is the aim of the authors to also emphasize those areas that require more research for a proper diagnosis and control of immunosuppressive diseases.

IMMUNOSUPPRESSIVE DISEASES OF POULTRY Isabel M. Gimeno Enrique Montiel Natàlia Majó Joan Smyth Arun K. R. Pandiri

• • • • •

Oscar J. Fletcher Orlando Osuna Roser Dolz Karel A. Schat Guillermo Zavala

INFECTIOUS BURSAL DISEASE

IMMUNOSUPPRESSIVE DISEASES OF POULTRY

Gross and microscopic lesions

Gross and microscopic lesions The most relevant gross lesion in IBDV infected animals is observed in the bursa of Fabricius, the target organ. In the early stages of the infection by classical or vvIBDV (2-3 dpi), a marked increase in size of the bursa due to acute inflammation can be observed. Edema of the bursal serosal surface is sometimes

Whereas at early stages the bursa a Fabricius increases in size due to inflammation and edema, the animals that survive after 8 days post-infection with classical and vvIBDV strains show bursal atrophy.

evident (Fig. 1) and small petechia in the mucosal surface of the bursa (Fig. 2) or extensive hemorrhages throughout the entire organ may be seen at this stage. At 5 to 8 dpi, animals that survive show marked bursal atrophy. 38 In addition, birds infected with classical or vvIBDV may show macroscopic lesions in other organs. Dehydration and hemorrhages in the thigh and pectoral muscles are very often observed (Fig. 3). Hemorrhages in the proventricular mucosa can also be seen, especially in vvIBDV-infected animals. Kidneys may also be secondarily affected, due to dehydration, and show paleness, a marked lobular pattern, and even urate deposition in the parenchyma and ureters (Fig. 4). 38 As regards

to other lymphoid organs, mild thymus atrophy 158 and mottling of the spleen 122 are the macroscopic lesions that have been more frequently described associated to classical and vvIBDV infection.

Experimental infection with variant E/Del IBDV strain resulted in bursal atrophy from 2 weeks post infection (wpi) that persisted until 6 wpi, without any evidence of acute inflammatory reaction. 31 Likewise, infection of four-day-old chickens with variant AL2 IBDV strain induced reduced bursal indices. 145 More relevant microscopic lesions in IBDV-infected animals are observed in the bursa of Fabricius. In the acute stage, lymphocytolysis of follicular medullary lymphocytes, associated to

a

b

500 µm

1 cm

Fig. 1 Marked peribursal edema in a broiler chicken with acute IBDV infection.

b

50 µm

74

Fig. 4 Severe nephropathy with marked intraparenchymal urate deposition, probably secondary to dehydration, in an IBDV-infected broiler chicken.

500 µm

Fig. 5 Microscopic image of a bursa of Fabricius from a broiler chicken with acute IBDV infection. (a) Diffuse lymphocytolysis associated to marked interfollicular edema and heterophilic and macrophagic influx is observed. HE staining. Bar = 500 μm. (b) Viral antigen widely distributed in follicular cells. Immunohistochemical staining with VP2 antibody. Bar = 500 μm.

Fig. 2 Multiple petechiae in the bursa of Fabricius in a broiler chicken with acute IBDV infection.

a

Fig. 3 Multiple petechiae and ecchymosis in the thigh muscles of a broiler chicken infected with IBDV.

marked interstitial edema, heterophilic and macrophage influx is the hallmark lesion of acute IBDV infection (Figs. 5a and 6a). Interstitial hemorrhages can also be frequently seen. Microscopic lesions tend to be diffuse, affecting all the follicles of the bursa and viral antigens are widely detected following immunohistochemical staining (Figs. 5b and 6b). At 4-5 dpi, a reduction in size of the lymphoid follicles with evident loss of follicular lymphocytes and replacement by macrophages containing cellular debris is observed. At more chronic stages, reduction of the size of the follicles due to lymphocyte depletion is more evident with shrinkage of the corticomedullary junction. Cystic cavities may develop in the medullary areas of the follicles, lined by a

50 µm

Fig. 6 Microscopic image of a bursa of Fabricius from a broiler chicken with acute IBDV infection. (a) Marked lymphocytolysis of follicular medullary lymphocytes associated to marked interfollicular edema and heterophilic and macrophagic influx. HE staining. Bar = 50 μm. (b) Viral antigen in the cytoplasm of follicular cells. Immunohistochemical staining with VP2 antibody. Bar = 50 μm.

75

INFECTIOUS BURSAL DISEASE

IMMUNOSUPPRESSIVE DISEASES OF POULTRY

Gross and microscopic lesions

Gross and microscopic lesions The most relevant gross lesion in IBDV infected animals is observed in the bursa of Fabricius, the target organ. In the early stages of the infection by classical or vvIBDV (2-3 dpi), a marked increase in size of the bursa due to acute inflammation can be observed. Edema of the bursal serosal surface is sometimes

Whereas at early stages the bursa a Fabricius increases in size due to inflammation and edema, the animals that survive after 8 days post-infection with classical and vvIBDV strains show bursal atrophy.

evident (Fig. 1) and small petechia in the mucosal surface of the bursa (Fig. 2) or extensive hemorrhages throughout the entire organ may be seen at this stage. At 5 to 8 dpi, animals that survive show marked bursal atrophy. 38 In addition, birds infected with classical or vvIBDV may show macroscopic lesions in other organs. Dehydration and hemorrhages in the thigh and pectoral muscles are very often observed (Fig. 3). Hemorrhages in the proventricular mucosa can also be seen, especially in vvIBDV-infected animals. Kidneys may also be secondarily affected, due to dehydration, and show paleness, a marked lobular pattern, and even urate deposition in the parenchyma and ureters (Fig. 4). 38 As regards

to other lymphoid organs, mild thymus atrophy 158 and mottling of the spleen 122 are the macroscopic lesions that have been more frequently described associated to classical and vvIBDV infection.

Experimental infection with variant E/Del IBDV strain resulted in bursal atrophy from 2 weeks post infection (wpi) that persisted until 6 wpi, without any evidence of acute inflammatory reaction. 31 Likewise, infection of four-day-old chickens with variant AL2 IBDV strain induced reduced bursal indices. 145 More relevant microscopic lesions in IBDV-infected animals are observed in the bursa of Fabricius. In the acute stage, lymphocytolysis of follicular medullary lymphocytes, associated to

a

b

500 µm

1 cm

Fig. 1 Marked peribursal edema in a broiler chicken with acute IBDV infection.

b

50 µm

74

Fig. 4 Severe nephropathy with marked intraparenchymal urate deposition, probably secondary to dehydration, in an IBDV-infected broiler chicken.

500 µm

Fig. 5 Microscopic image of a bursa of Fabricius from a broiler chicken with acute IBDV infection. (a) Diffuse lymphocytolysis associated to marked interfollicular edema and heterophilic and macrophagic influx is observed. HE staining. Bar = 500 μm. (b) Viral antigen widely distributed in follicular cells. Immunohistochemical staining with VP2 antibody. Bar = 500 μm.

Fig. 2 Multiple petechiae in the bursa of Fabricius in a broiler chicken with acute IBDV infection.

a

Fig. 3 Multiple petechiae and ecchymosis in the thigh muscles of a broiler chicken infected with IBDV.

marked interstitial edema, heterophilic and macrophage influx is the hallmark lesion of acute IBDV infection (Figs. 5a and 6a). Interstitial hemorrhages can also be frequently seen. Microscopic lesions tend to be diffuse, affecting all the follicles of the bursa and viral antigens are widely detected following immunohistochemical staining (Figs. 5b and 6b). At 4-5 dpi, a reduction in size of the lymphoid follicles with evident loss of follicular lymphocytes and replacement by macrophages containing cellular debris is observed. At more chronic stages, reduction of the size of the follicles due to lymphocyte depletion is more evident with shrinkage of the corticomedullary junction. Cystic cavities may develop in the medullary areas of the follicles, lined by a

50 µm

Fig. 6 Microscopic image of a bursa of Fabricius from a broiler chicken with acute IBDV infection. (a) Marked lymphocytolysis of follicular medullary lymphocytes associated to marked interfollicular edema and heterophilic and macrophagic influx. HE staining. Bar = 50 μm. (b) Viral antigen in the cytoplasm of follicular cells. Immunohistochemical staining with VP2 antibody. Bar = 50 μm.

75

IMMUNOSUPPRESSIVE DISEASES OF POULTRY

MAREK’S DISEASE VIRUS INFECTION AND ASSOCIATED SYNDROMES MDV-induced syndromes

Gross lesions: bursa of Fabricius and thymus are atrophic and yellowish as early as 6-8 dpi (Fig. 4a). The spleen is enlarged and in many cases marbled at 4-6 dpi. After infection with highly virulent MDV (vv and vv+ MDV), necrotic foci can be observed mainly in the spleen at about 7-10 dpi. 114 Necrosis in lymphoid organs is more evident when 1-day-old susceptible chickens are inoculated with a highly virulent MDV. Histopathology: the productive infection in lymphoid organs reaches a peak at 5-6 dpi and it is accompanied by cytolysis of lymphocytes and other cells, hyperplasia of reticular cells, accumulation of macrophages and infiltration by granulocytes. In the thymus, the cytolytic infection and hyperplasia of reticular cells occur mainly in the medulla, and the cortex is markedly atrophied (Fig. 5a). Similar changes occur in the bursa, with destruction of the normal architecture (Fig. 5b). When atrophy of the bursa and thymus is not reversible, connective tissue replaces the parenchyma and the lumen is lined by unfolded/undulating epithelium (Figs. 4b-c).

Immunohistochemistry: early cytolytic infection in lymphoid organs is characterized by the production of abundant viral antigen (both early and late viral antigens), as can be detected by a variety of techniques. Most of the antigens in the bursa and thymus can be detected in the medulla where necrosis is evident (Figs. 5c-d). In severe cases, viral antigens can be detected in both medulla and cortex (Figs. 5e-f).

Diagnosis: lymphodegenerative syndromes do not occur in chickens vaccinated against MD or in chickens bearing MAb. Therefore, it is a highly uncommon scenario under field conditions. In unvaccinated chickens, MDV could induce lymphoid organ atrophy once MAb levels decrease (around 3 weeks of age).

In those cases, diagnosis of lymphodegenerative syndromes is very difficult and a variety of other diseases of infectious (infectious bursal disease, chicken infectious anemia, etc.) and noninfectious (aflatoxins, stress) etiology have to be included in the differential diagnosis as all of them could induce necrosis and atrophy of the lymphoid organs. Detection of MDV antigens in the lymphoid organs has very little diagnostic value because it is transient (5-6 dpi) and occurs before the lymphoid organ atrophy is apparent.

Control: vaccination with any of the currently available MD vaccines protects against the development of lymphodegenerative syndromes.

a

b

c

Fig. 3 Transient paralysis (TP). (a) Chicken showing flaccid neck paralysis that is characteristic of TP. (b) Chicken showing depression and limb paraly­ sis. Please note that this chicken (10 days old) still maintains baby fea­ thers due to delayed maturation secondary to viral infection; (c) Cerebel­ lum: vasculitis and perivascular edema are characteristic of TP.

128

Early inflammatory lesions in other tissues Field relevance: MDV induces inflammatory and degenerative lesions in various organs that are not accompanied by the de-

velopment of clinical signs and that may or may not develop into neoplastic lesions (Figs. 6a-d). It is important to differentiate between those inflammatory lesions (indication of MDV infection) and neoplastic lesions (indication of MD).

Clinical signs: none.

Gross lesions: none.

Histopathology: inflammatory infiltrates in the nerves (Fig. 6a) can occur very early after MDV infection (7-8 dpi) and is of particular relevance due to the confusions in diagnosis. Mild inflammatory lesions in the nerves can be induced by all three MDV serotypes and they can regress without inducing clinical signs. It is important that these inflammatory lesions do not get confused with lymphomas in nerves. This is also true for the presence of mononuclear perivascular cuffing in the brain that is not associated with vasculitis and edema (Fig. 6b). Those lesions are common in chickens infected with MDV but are not related to any disease.

Early lesions in skin are not normally associated with clinical signs or gross lesions such as enlarged feather follicles and in general have to be identified by microscopic analysis. They are important as they seem to be necessary for the infection of the FFE and therefore for the transmission of the infection. Early pathological changes in the skin include dermatitis (perifollicular and follicular) as well as degeneration in the FFE due to the repli­ cation of MDV. Diagnosis: the relevance of these inflammatory lesions is presented in the differential diagnosis with tumors induced by MDV. Control: inflammatory lesions are a common finding in chickens infected with MDV regardless of their health status. MDV is ubiquitous and except those flocks maintained under specific pathogen free (SPF) conditions, commercial flocks will invariably be infected with MDV and chickens will have MDV-induced inflammatory lesions, with or without MD. The presence of MDV-induced inflammatory lesions alone does not support a diagnosis of MD but just of MDV infection. Furthermore, MDV-induced inflammatory lesions in chickens with tumors due to other etiologies might complicate the diagnosis and in these cases additional tests will be required for confirming the diagnosis.

a

b

c

Panophthalmitis and ocular disease Fig. 4 Lymphoid organ atrophy associated with MDV infection. (a) The bursa of Fabricius (left) and two thymic lobes (right) in the upper row are from healthy uninfected chickens. Severely atrophied bursa of Fabricius and two thymic lobes of chickens 10 days after inoculation with very virulent plus (vv+) MDV strain 648A can be observed in the low row. (b) Photo­ micrograph of a severely atrophied bursa of Fabricius (hematoxylin and eosin stain). (c) Photomicrograph of a severely atrophied thymic lobe (hematoxylin and eosin stain).

Field relevance: the relevance of ocular lesions in the field is unknown. Increased ocular lesions have been associated with outbreaks induced by highly virulent MDV (vv and vv+ MDV). 39,104 Vaccination does not always result in protection against ocular lesions. 82 The progression in the severity of the ocular lesions undoubtedly will result in blindness. However, the economic 129

IMMUNOSUPPRESSIVE DISEASES OF POULTRY

MAREK’S DISEASE VIRUS INFECTION AND ASSOCIATED SYNDROMES MDV-induced syndromes

Gross lesions: bursa of Fabricius and thymus are atrophic and yellowish as early as 6-8 dpi (Fig. 4a). The spleen is enlarged and in many cases marbled at 4-6 dpi. After infection with highly virulent MDV (vv and vv+ MDV), necrotic foci can be observed mainly in the spleen at about 7-10 dpi. 114 Necrosis in lymphoid organs is more evident when 1-day-old susceptible chickens are inoculated with a highly virulent MDV. Histopathology: the productive infection in lymphoid organs reaches a peak at 5-6 dpi and it is accompanied by cytolysis of lymphocytes and other cells, hyperplasia of reticular cells, accumulation of macrophages and infiltration by granulocytes. In the thymus, the cytolytic infection and hyperplasia of reticular cells occur mainly in the medulla, and the cortex is markedly atrophied (Fig. 5a). Similar changes occur in the bursa, with destruction of the normal architecture (Fig. 5b). When atrophy of the bursa and thymus is not reversible, connective tissue replaces the parenchyma and the lumen is lined by unfolded/undulating epithelium (Figs. 4b-c).

Immunohistochemistry: early cytolytic infection in lymphoid organs is characterized by the production of abundant viral antigen (both early and late viral antigens), as can be detected by a variety of techniques. Most of the antigens in the bursa and thymus can be detected in the medulla where necrosis is evident (Figs. 5c-d). In severe cases, viral antigens can be detected in both medulla and cortex (Figs. 5e-f).

Diagnosis: lymphodegenerative syndromes do not occur in chickens vaccinated against MD or in chickens bearing MAb. Therefore, it is a highly uncommon scenario under field conditions. In unvaccinated chickens, MDV could induce lymphoid organ atrophy once MAb levels decrease (around 3 weeks of age).

In those cases, diagnosis of lymphodegenerative syndromes is very difficult and a variety of other diseases of infectious (infectious bursal disease, chicken infectious anemia, etc.) and noninfectious (aflatoxins, stress) etiology have to be included in the differential diagnosis as all of them could induce necrosis and atrophy of the lymphoid organs. Detection of MDV antigens in the lymphoid organs has very little diagnostic value because it is transient (5-6 dpi) and occurs before the lymphoid organ atrophy is apparent.

Control: vaccination with any of the currently available MD vaccines protects against the development of lymphodegenerative syndromes.

a

b

c

Fig. 3 Transient paralysis (TP). (a) Chicken showing flaccid neck paralysis that is characteristic of TP. (b) Chicken showing depression and limb paraly­ sis. Please note that this chicken (10 days old) still maintains baby fea­ thers due to delayed maturation secondary to viral infection; (c) Cerebel­ lum: vasculitis and perivascular edema are characteristic of TP.

128

Early inflammatory lesions in other tissues Field relevance: MDV induces inflammatory and degenerative lesions in various organs that are not accompanied by the de-

velopment of clinical signs and that may or may not develop into neoplastic lesions (Figs. 6a-d). It is important to differentiate between those inflammatory lesions (indication of MDV infection) and neoplastic lesions (indication of MD).

Clinical signs: none.

Gross lesions: none.

Histopathology: inflammatory infiltrates in the nerves (Fig. 6a) can occur very early after MDV infection (7-8 dpi) and is of particular relevance due to the confusions in diagnosis. Mild inflammatory lesions in the nerves can be induced by all three MDV serotypes and they can regress without inducing clinical signs. It is important that these inflammatory lesions do not get confused with lymphomas in nerves. This is also true for the presence of mononuclear perivascular cuffing in the brain that is not associated with vasculitis and edema (Fig. 6b). Those lesions are common in chickens infected with MDV but are not related to any disease.

Early lesions in skin are not normally associated with clinical signs or gross lesions such as enlarged feather follicles and in general have to be identified by microscopic analysis. They are important as they seem to be necessary for the infection of the FFE and therefore for the transmission of the infection. Early pathological changes in the skin include dermatitis (perifollicular and follicular) as well as degeneration in the FFE due to the repli­ cation of MDV. Diagnosis: the relevance of these inflammatory lesions is presented in the differential diagnosis with tumors induced by MDV. Control: inflammatory lesions are a common finding in chickens infected with MDV regardless of their health status. MDV is ubiquitous and except those flocks maintained under specific pathogen free (SPF) conditions, commercial flocks will invariably be infected with MDV and chickens will have MDV-induced inflammatory lesions, with or without MD. The presence of MDV-induced inflammatory lesions alone does not support a diagnosis of MD but just of MDV infection. Furthermore, MDV-induced inflammatory lesions in chickens with tumors due to other etiologies might complicate the diagnosis and in these cases additional tests will be required for confirming the diagnosis.

a

b

c

Panophthalmitis and ocular disease Fig. 4 Lymphoid organ atrophy associated with MDV infection. (a) The bursa of Fabricius (left) and two thymic lobes (right) in the upper row are from healthy uninfected chickens. Severely atrophied bursa of Fabricius and two thymic lobes of chickens 10 days after inoculation with very virulent plus (vv+) MDV strain 648A can be observed in the low row. (b) Photo­ micrograph of a severely atrophied bursa of Fabricius (hematoxylin and eosin stain). (c) Photomicrograph of a severely atrophied thymic lobe (hematoxylin and eosin stain).

Field relevance: the relevance of ocular lesions in the field is unknown. Increased ocular lesions have been associated with outbreaks induced by highly virulent MDV (vv and vv+ MDV). 39,104 Vaccination does not always result in protection against ocular lesions. 82 The progression in the severity of the ocular lesions undoubtedly will result in blindness. However, the economic 129

RETICULOENDOTHELIOSIS AND OTHER IMMUNOSUPPRESSIVE VIRUSES

IMMUNOSUPPRESSIVE DISEASES OF POULTRY

Diagnosis

120.0

100.0

Cumulative Mortality (%)

80.0

60.0 PM REV + PM

40.0

REV + Vaccine + PM Vaccine + PM

20.0

0.0 5:00 PM

9:00 AM 1

9:00 AM

5:00 PM

9:00 AM

5:00 PM

2

9:00 AM

3

5:00 PM

9:00 AM

4

5:00 PM 5

Days and Time Post-Inoculation

Fig. 5 Death time induced by Pasteurella multocida in REV-free and REV-infected SPF turkeys. Only relevant experimental groups are shown: PM = P. multocida only; REV+PM = P. multocida challenge + REV-infected; REV + Vaccine + PM = REV-infected + P. multocida bacterin + P. multocida challenge; Vaccine + PM = P. multocida bacterin + P. multocida challenge. The first turkeys to die after a virulent PM challenge were those infected with REV+PM in the absence of a bacterin; followed by turkeys challenged with PM without a bacterin; followed by vaccinated turkeys, infected with REV and challenged with PM; and finally turkeys vaccinated and challenged with PM without REV infection. Modified from: Barbosa et al., Virus Res. 124:68-77. 2007.

162

190.0 180.0 170.0 AVG Body Weight (g) at 17D

SPF chickens co-infected with REV and NDV, as opposed to chickens infected with NDV alone. 154 Besides the reduction of immune competency against the bacterium Pasteurella multocida, decreased immune responses against other bacterial species such as Salmonella typhimurium (ST) have been documented. Deaths attributable to ST were higher in chickens co-infected with REV and ST than in chickens infected with ST alone. 88 Increased severity of clinical signs, lesions, and mortality has been reported in chickens co-infected with infectious laryngotracheitis virus and REV and in birds co-infected with REV and fowl poxvirus. 50,90 Experimental and natural infections with REV and ALV have been reported. 47 Co-infection with REV and ALV results in a higher frequency of ALV viremic chickens than in populations infected with ALV alone. In addition, chickens co-infected with REV and ALV responded less efficiently with sensitization against “symbolic” antigens such as sheep red blood cells (SRBC) and Brucella abortus. Furthermore, the strain used in such experiments, REV-T, induced a marked reduction of the bursa of

160.0 150.0 140.0 130.0 120.0 110.0

Fabricius in commercial chickens. 47 Natural outbreaks with simultaneous infection with REV and ALV subgroup J (ALV-J) apparently facilitated vertical transmission of ALV-J and enhanced the expression of myelocytomas in commercial meat type and egg type chickens of China, including indigenous Chinese breeds. 27 REV can interact with other immunosuppressive viruses such as MDV and CIAV, as has been shown experimentally and in natural outbreaks. REV-specific cytotoxic T cells are not responsive when chickens devoid of antibodies against CIAV are infected with CIAV at 9-17 days of age. 80 Immunity to MD vaccines is significantly reduced in chickens infected with REV. 145 An additional and unusual form of viral interaction may occur from REV inserting fragments of its own genome or the complete REV genome into the genomes of large DNA viruses such as MDV and poxviruses, or simply by contamination of MD or fowl poxvirus vaccines, which may modify the virulence of MDV or poxviruses and the outcome of infection by any such viruses. 4,15,28,34,48,49,53,60,62,69,79,86,90,104,119-121,129-131,139,146 Other forms of viral interactions exist and do not necessarily result in enhanced immunosuppression. In one experiment, a turkey strain designated as REV 397-A and isolated from a natural outbreak of RE in commercial turkeys was co-inoculated with turkey coronavirus (TCoV-TXp7), kindly provided by Dr. Mark Jackwood, University of Georgia. Turkeys co-infected with REV and turkey coronavirus expressed lower body weights than turkeys inoculated with either one of the viruses alone, suggesting that REV can interact in more ways than just immunosuppression (Fig. 6). Co-infection with TCoV and REV extended the period of detectable REV viremia and also the proportion of REV viremic turkeys in the experiment. Finally, the bursal microscopic lesions representing lymphocytic depletion were more severe in the turkeys that received both viruses instead of either one alone (Turner, Zavala, Barbosa and Cheng, unpublished).

REV infection can result in enhanced frequency and severity of lesions associated with Eimeria tenella 89, indicating that REV not only compromises responses against other viruses but to protozoa as well, which is expected since cellular immunity is clearly compromised. 21,22,55-58

100.0 Control

REV

TCoV

REV + TCoV

Fig. 6 Interactions between REV and a non-neoplastic, non-immunosuppressive virus (turkey coronavirus, TCoV). Dual infection with REV and TCoV resulted in enhanced depression at 17 days of age in experimentallyinfected SPF turkeys.

Economic effects Other than in commercial chickens of China, clinical RE occurs only sporadically elsewhere in the world. Field infection in adult chickens and turkeys is evidenced by frequent seroconversion

observed in commercial flocks, rarely with any evidence of clinical disease, mortality or virus shedding into fertile eggs. The exception has been occasional outbreaks associated with contaminated vaccines against MD or fowl poxvirus. 49,62,72,73 In those cases, economic losses have been rather dramatic and resulted from high mortality with tumors, delayed growth, a need for depopulating broiler breeder flocks and in some cases, litigation. Increased mortality associated with tumors, increased condemnations and decreased egg production have been the most relevant detrimental effects, as it occurred in one of the most recent natural outbreaks of RE in commercial turkeys. 26 Producers of SPF eggs and chickens, vaccine manufacturing companies and producers of meat and egg type breeding stock must invest in costly REV surveillance, as required by law and by importers of breeding stock such as Israel.

In an attempt to further define the economic impact of REV infection, a disease model was pursued in Japanese quail. The effects of RE were studied in two consecutive generations of quail. Delayed growth and increased mortality with tumors were detected by 6-8 weeks of age. Egg production, hatchability and fertility rates were reduced in REV-infected quail in comparison with uninfected quail. Body weight gain was significantly reduced by 8 weeks of age in the first generation of infected quail (quail breeders) and at 3 and 6 weeks of age in the second generation in contrast with non-infected quail (Fig. 6). 128

Interactions with other Infectious Agents As indicated above, REV may interact with other immunosuppressive agents, including MDV, CIAV and IBDV, and with nonimmunosuppressive viruses, bacteria, fungi and protozoa (see section on Immune responses to avian pathogens).

Diagnosis Diagnosis of neoplastic diseases can be done in a targeted fashion or by exclusion. That is, by excluding other oncogenic viruses using appropriate diagnostic techniques. REV does not have an endogenous virus counterpart as ALV does and there are no live or killed vaccines against REV that would be detectable by virus isolation, antigen capture ELISA or molecular methods. Antibody ELISA kits are available commercially for detection of seroconversion.

163

RETICULOENDOTHELIOSIS AND OTHER IMMUNOSUPPRESSIVE VIRUSES

IMMUNOSUPPRESSIVE DISEASES OF POULTRY

Diagnosis

120.0

100.0

Cumulative Mortality (%)

80.0

60.0 PM REV + PM

40.0

REV + Vaccine + PM Vaccine + PM

20.0

0.0 5:00 PM

9:00 AM 1

9:00 AM

5:00 PM

9:00 AM

5:00 PM

2

9:00 AM

3

5:00 PM

9:00 AM

4

5:00 PM 5

Days and Time Post-Inoculation

Fig. 5 Death time induced by Pasteurella multocida in REV-free and REV-infected SPF turkeys. Only relevant experimental groups are shown: PM = P. multocida only; REV+PM = P. multocida challenge + REV-infected; REV + Vaccine + PM = REV-infected + P. multocida bacterin + P. multocida challenge; Vaccine + PM = P. multocida bacterin + P. multocida challenge. The first turkeys to die after a virulent PM challenge were those infected with REV+PM in the absence of a bacterin; followed by turkeys challenged with PM without a bacterin; followed by vaccinated turkeys, infected with REV and challenged with PM; and finally turkeys vaccinated and challenged with PM without REV infection. Modified from: Barbosa et al., Virus Res. 124:68-77. 2007.

162

190.0 180.0 170.0 AVG Body Weight (g) at 17D

SPF chickens co-infected with REV and NDV, as opposed to chickens infected with NDV alone. 154 Besides the reduction of immune competency against the bacterium Pasteurella multocida, decreased immune responses against other bacterial species such as Salmonella typhimurium (ST) have been documented. Deaths attributable to ST were higher in chickens co-infected with REV and ST than in chickens infected with ST alone. 88 Increased severity of clinical signs, lesions, and mortality has been reported in chickens co-infected with infectious laryngotracheitis virus and REV and in birds co-infected with REV and fowl poxvirus. 50,90 Experimental and natural infections with REV and ALV have been reported. 47 Co-infection with REV and ALV results in a higher frequency of ALV viremic chickens than in populations infected with ALV alone. In addition, chickens co-infected with REV and ALV responded less efficiently with sensitization against “symbolic” antigens such as sheep red blood cells (SRBC) and Brucella abortus. Furthermore, the strain used in such experiments, REV-T, induced a marked reduction of the bursa of

160.0 150.0 140.0 130.0 120.0 110.0

Fabricius in commercial chickens. 47 Natural outbreaks with simultaneous infection with REV and ALV subgroup J (ALV-J) apparently facilitated vertical transmission of ALV-J and enhanced the expression of myelocytomas in commercial meat type and egg type chickens of China, including indigenous Chinese breeds. 27 REV can interact with other immunosuppressive viruses such as MDV and CIAV, as has been shown experimentally and in natural outbreaks. REV-specific cytotoxic T cells are not responsive when chickens devoid of antibodies against CIAV are infected with CIAV at 9-17 days of age. 80 Immunity to MD vaccines is significantly reduced in chickens infected with REV. 145 An additional and unusual form of viral interaction may occur from REV inserting fragments of its own genome or the complete REV genome into the genomes of large DNA viruses such as MDV and poxviruses, or simply by contamination of MD or fowl poxvirus vaccines, which may modify the virulence of MDV or poxviruses and the outcome of infection by any such viruses. 4,15,28,34,48,49,53,60,62,69,79,86,90,104,119-121,129-131,139,146 Other forms of viral interactions exist and do not necessarily result in enhanced immunosuppression. In one experiment, a turkey strain designated as REV 397-A and isolated from a natural outbreak of RE in commercial turkeys was co-inoculated with turkey coronavirus (TCoV-TXp7), kindly provided by Dr. Mark Jackwood, University of Georgia. Turkeys co-infected with REV and turkey coronavirus expressed lower body weights than turkeys inoculated with either one of the viruses alone, suggesting that REV can interact in more ways than just immunosuppression (Fig. 6). Co-infection with TCoV and REV extended the period of detectable REV viremia and also the proportion of REV viremic turkeys in the experiment. Finally, the bursal microscopic lesions representing lymphocytic depletion were more severe in the turkeys that received both viruses instead of either one alone (Turner, Zavala, Barbosa and Cheng, unpublished).

REV infection can result in enhanced frequency and severity of lesions associated with Eimeria tenella 89, indicating that REV not only compromises responses against other viruses but to protozoa as well, which is expected since cellular immunity is clearly compromised. 21,22,55-58

100.0 Control

REV

TCoV

REV + TCoV

Fig. 6 Interactions between REV and a non-neoplastic, non-immunosuppressive virus (turkey coronavirus, TCoV). Dual infection with REV and TCoV resulted in enhanced depression at 17 days of age in experimentallyinfected SPF turkeys.

Economic effects Other than in commercial chickens of China, clinical RE occurs only sporadically elsewhere in the world. Field infection in adult chickens and turkeys is evidenced by frequent seroconversion

observed in commercial flocks, rarely with any evidence of clinical disease, mortality or virus shedding into fertile eggs. The exception has been occasional outbreaks associated with contaminated vaccines against MD or fowl poxvirus. 49,62,72,73 In those cases, economic losses have been rather dramatic and resulted from high mortality with tumors, delayed growth, a need for depopulating broiler breeder flocks and in some cases, litigation. Increased mortality associated with tumors, increased condemnations and decreased egg production have been the most relevant detrimental effects, as it occurred in one of the most recent natural outbreaks of RE in commercial turkeys. 26 Producers of SPF eggs and chickens, vaccine manufacturing companies and producers of meat and egg type breeding stock must invest in costly REV surveillance, as required by law and by importers of breeding stock such as Israel.

In an attempt to further define the economic impact of REV infection, a disease model was pursued in Japanese quail. The effects of RE were studied in two consecutive generations of quail. Delayed growth and increased mortality with tumors were detected by 6-8 weeks of age. Egg production, hatchability and fertility rates were reduced in REV-infected quail in comparison with uninfected quail. Body weight gain was significantly reduced by 8 weeks of age in the first generation of infected quail (quail breeders) and at 3 and 6 weeks of age in the second generation in contrast with non-infected quail (Fig. 6). 128

Interactions with other Infectious Agents As indicated above, REV may interact with other immunosuppressive agents, including MDV, CIAV and IBDV, and with nonimmunosuppressive viruses, bacteria, fungi and protozoa (see section on Immune responses to avian pathogens).

Diagnosis Diagnosis of neoplastic diseases can be done in a targeted fashion or by exclusion. That is, by excluding other oncogenic viruses using appropriate diagnostic techniques. REV does not have an endogenous virus counterpart as ALV does and there are no live or killed vaccines against REV that would be detectable by virus isolation, antigen capture ELISA or molecular methods. Antibody ELISA kits are available commercially for detection of seroconversion.

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