Liver Pathology Associated with Increased Mortality in Turkey Breeder and Meat Turkey Flocks Author(s): Christina Popp, Rüdiger Hauck, Thomas W. Vahlenkamp, Dörte Lüschow, Olivia Kershaw, Marc Hoferer, and Hafez M. Hafez Source: Avian Diseases, 58(3):474-481. 2014. Published By: American Association of Avian Pathologists DOI: http://dx.doi.org/10.1637/10675-092413-Case.1 URL: http://www.bioone.org/doi/full/10.1637/10675-092413-Case.1
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AVIAN DISEASES 58:474–481, 2014
Case Report— Liver Pathology Associated with Increased Mortality in Turkey Breeder and Meat Turkey Flocks Christina Popp,A Ru¨diger Hauck,BF Thomas W. Vahlenkamp,C Do¨rte Lu¨schow,B Olivia Kershaw,D Marc Hoferer,E and Hafez M. HafezBG A Poultry Health Service Baden-Wu¨rttemberg, Schaflandstr. 3/3, 70736 Fellbach, Germany Institute for Poultry Diseases, Freie Universita¨t Berlin, Ko¨nigsweg 63, 14163 Berlin, Germany C Institute of Virology, University of Leipzig, An den Tierkliniken 29, 04103 Leipzig, Germany D Department of Veterinary Pathology, Freie Universita¨t Berlin, Robert-von-Ostertag-Strasse 15, 14163 Berlin, Germany E Chemical and Veterinary Investigations Office, Stuttgart (CVUAS), Schaflandstr. 3/3, 70736 Fellbach, Germany B
Received 1 October 2013; Accepted 17 February 2014; Published ahead of print 17 February 2014 SUMMARY. Between 2006 and 2011 a series of disease conditions characterized by raised mortality and liver disorders occurred in turkey breeder flocks and in meat turkey flocks in Germany. The flocks were between 12 and 23 wk of age, and mostly hens were affected. Clinical signs were nonspecific and accompanied by mortality varying between 1% and 7%. Affected birds displayed swollen livers that were marbled with black and red spots and yellowish areas. The pericardium was filled with an amber fluid, and the coronary groove was extensively filled with fat. Spleens were swollen, and a serous fluid that seemed to leak from the liver was present in the body cavity. Histopathological findings in all but one case included fatty degeneration of hepatocytes with parenchymal collapse and associated hemorrhages. Some animals showed cholangitis and hepatitis with intranuclear inclusion bodies. In three cases with breeders, electron microscopy detected virus particles that were between 23 and 30 nm and similar to parvo- or picornavirus. In addition, picornavirus RNA was detected in the livers of one meat turkey flock. Investigations by PCR for circovirus, polyomavirus parvovirus, and aviadenovirus yielded negative results in all cases, but an aviadenovirus was isolated from livers twice and a reovirus from the intestines once. Supplementation with vitamin E and selenium seemed to improve the situation. The most likely diagnosis is lipidosis, a metabolic disorder with complex etiology, which has rarely been described in turkeys. RESUMEN. Reporte de Caso—Patologı´a hepa´tica asociada con aumento de mortalidad en parvadas de pavos reproductores y de engorde. Entre 2006 y 2011 se presentaron una serie de enfermedades caracterizadas por aumento de la mortalidad y problemas hepa´ticos en parvadas de pavos reproductores y de engorde en Alemania. Las parvadas tenı´an entre 12 y 23 semanas de edad y en su mayorı´a las gallinas fueron las ma´s afectadas. Los signos clı´nicos fueron inespecı´ficos y estuvieron acompan˜ados por mortalidad que oscilo´ entre el 1% y 7%. Las aves afectadas mostraron hı´gados inflamados que presentaban aspecto marmoleado con a´reas amarillentas y puntos negros y rojos. El pericardio estaba lleno de un fluido de color a´mbar, y el surco coronario estaba extensivamente lleno de grasa. Los bazos se observaron inflamados y un fluido seroso que parecı´a salir del hı´gado, estaba presente en la cavidad corporal. Los hallazgos histopatolo´gicos en todos los casos menos en uno incluyen degeneracio´n grasa de los hepatocitos con colapso del pare´nquima y hemorragias asociadas. Algunos animales mostraron colangitis y hepatitis con cuerpos de inclusio´n intranucleares. En tres casos con reproductores, la microscopı´a electro´nica detecto´ partı´culas virales que oscilaban entre 23 y 30 nm, similares a parvo o picornavirus. Adema´s, se detecto´ ARN de picornavirus en los hı´gados de una parvada de pavos de engorde. Las investigaciones por PCR para circovirus, parvovirus, poliomavirus y aviadenovirus mostraron resultados negativos en todos los casos, pero se aislo´ un aviadenovirus a partir de hı´gados dos veces y un reovirus de los intestinos en una vez. La suplementacio´n con vitamina E y el selenio parecieron mejorar la situacio´n. El diagno´stico ma´s probable fue lipidosis, un trastorno metabo´lico con etiologı´a compleja, que rara vez se ha descrito en pavos. Key words: turkeys, metabolic disorders, lipidosis Abbreviations: CE 5 chicken embryo; DMEM 5 Dulbecco’s modified Eagle’s medium; HE 5 hematoxylin and eosin; PBS 5 phosphate-buffered saline; RT 5 reverse transcription
The liver is the main metabolic organ of birds. Among its tasks are detoxication and formation of blood proteins. It is also an important site of lipid metabolism. Diseases of the liver in poultry can be caused by infectious or noninfectious causes. Among the numerous pathogens causing liver lesions are viruses (aviadenoviruses (1), picornavirus (16), tumor viruses (7,30)), bacteria (Escherichia coli F Present address: Federal Office for Consumer Protection and Food Safety, Mauerstr. 39-42, 10117 Berlin, Germany G Corresponding author. E-mail: [email protected]
(3), Clostridium perfringens (6,21,23), Chlamydia psittaci (2)), and the protozoan parasite Histomonas meleagridis (20). Noninfectious causes include intoxications, especially with mycotoxins (15), and metabolic disorders like hepatic lipidosis (4,8). During the last several years a series of disease conditions characterized by liver disorders and accompanied by increased mortality occurred in turkey breeder flocks and in meat turkey flocks in Germany. Here we describe several cases that occurred in the state of Baden-Wu¨rttemberg in Southern Germany between 2006 and 2011.
474
475
Liver changes in turkey flocks
Fig. 1.
Typical liver appearance in diseased birds in two cases in breeder hens (A) and meat turkey hens (B).
MATERIALS AND METHODS Microbiology. Samples from heart blood, livers, and lungs were collected with sterile disposable bacterial loops and streaked on Columbia agar containing 7% sheep blood and Gassner agar (Oxoid, Wesel, Germany). Plates were incubated at 37uC overnight under aerobic conditions. Histopathology. Liver samples were fixed in 4% formalin and embedded in paraffin following standard laboratory procedures. Sections (3 mm) of dewaxed tissues were stained with hematoxylin and eosin (HE) for histopathological investigation. Electron microscopy. After negative staining, transmission electron microscopy (80 keV microscope JEM-1011; Jeol, Eching, Germany) was used to identify viral particles in livers. One grid per sample was prepared. Briefly, after the grids were cleaned in an ultrasonic bath using acetic acid and washed, they were coated with a 0.8% solution of pioloform (PLANO, Wetzlar, Germany) and ROTIPURANH chloroform (Roth, Karlsruhe, Germany). After cell disruption of the tissue samples (50 mg of tissue in 500 ml of double-distilled water) with a TeSeE PRECESSH 24 homogenizer (Bio-Rad, Munich, Germany), the samples were centrifuged at 420 3 g for 10 min, and the supernatants transferred onto a Parafilm ‘‘M’’ (Brand, Wertheim, Germany). Subsequently, the glow discharge-pretreated carbon grids were coated with the different samples. Phosphotungstic acid solution (1% [w/v], pH 7.2) was used for negative staining. Images (200,0003 amplification) were taken with the SIS Megaview III CCD Olympus camera. The software AnalySIS (Olympus, Mu¨nster, Germany) was used to take size measurements of the viruses and to identify typical structural criteria mentioned in the literature (5,14,18). Virus isolation. Chicken embryo (CE) fibroblasts and CE liver cells were prepared from 10-day-old embryos according to standard protocols (24). Cells were grown as monolayer until near confluence was obtained. Primary and secondary cultures of CE cells were maintained at 38 C and 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% fetal calf serum, 50 U/ml penicillin, and 50 mg/ml streptomycin. For infection experiments, CE cells were washed with phosphate-buffered saline (PBS) and incubated with organ supernatants. These were obtained by mincing 1 g of organ material with sand and 10 ml of PBS containing 100 U/ml penicillin and 100 mg/ml streptomycin in a mortar under sterile conditions and subsequent incubation for 30 min at room temperature. After two freeze–thaw cycles at 280 C the virus containing supernatant was harvested after centrifugation at 1500 3 g for 5 min. The supernatant was filter sterilized with a 0.20-mm filter, and 500 ml was used for each well of a 24-well plate. All infections were performed in duplicate. Mock-infected cells were used as controls. After incubation with the supernatants for 60 min, CE cells were rinsed and DMEM was added as already described. Supernatants were harvested when a cytopathic effect had developed, usually at 3 days after infection. Polymerase chain reaction. The isolation of nucleic acids from organ samples was performed by mincing 25 mg of the organ material with sand under sterile conditions following centrifugation at room temperature at
1048 3 g for 5 min. The supernatants of the organ suspensions were subjected to DNA or RNA isolation using the DNeasy and RNeasy Mini Kit (Qiagen, Hilden, Germany), respectively. Five microliters of the purified nucleic acids was used for the subsequent amplification. The presence of avian circoviruses was analyzed according to Halami et al. (11) using a consensus nested PCR, which enables the detection of a broad spectrum of avian circoviruses. The nested PCR amplified a product of 350 bp in length. Polyomavirus was detected according to Johne et al. (17) using a consensus nested PCR, which enables the detection of a broad spectrum of polyomaviruses in different species. The nested PCR amplified a 273bp product. The presence of aviadenovirus was analyzed using PCR, which amplified a product of 590 bp (22). Parvovirus was analyzed according to Zsak et al. (32). The PCR generated a product of 561 bp in length. Avian reovirus was detected according to Wellehan et al. (29) using a consensus reverse transcription (RT) nested PCR targeting conserved regions of the viral polymerase gene. The RT-PCR generated a 226 bpproduct. Avian picornavirus was detected according to Xie et al. (31) using primers targeting the VP2 gene of avian encephalomyelitis virus. The RT step generated a 619-bp cDNA product, which was subsequently amplified.
CASE REPORT
The initial case. The disease was first observed in 2006 in a flock of 5250 turkey breeder hens. At the age of 22 to 23 wk mortality started to rise. Initially about four to six birds died each day, but the number of daily fatalities increased up to 80 birds a day. Within 2 wk, 5.3% of the birds died. Affected birds withdrew from the other birds, laid down, closed their eyes, dropped their heads, flapped their wings, and gasped. Furthermore, they bled out of the beak. Birds died within 2–6 hr after showing these symptoms. At postmortem, livers were swollen, marbled with black and red spots, and showed yellow areas (Fig. 1). The pericardium was filled with an amber fluid, the coronary groove was extensively filled with fat, and spleens were swollen. A blood-tinged fluid was in the abdominal cavity due to liver rupture. Furthermore, catarrhal enteritis was observed. Escherichia coli was isolated from the hearts, livers, and lungs of some birds. Histopathological investigation identified a severe hepatic dystrophy with severe fatty degeneration and centrilobular necroses, disseminated hemorrhages, and periportal infiltrations (Fig. 2). Eosinophilic intranuclear inclusion bodies suggested a viral infection (Fig. 3). Centrifollicular and perivascular fibrinous exudates admixed with extracellular and intrahistiocytic bacteria were present in the spleen. Furthermore, a few necrotic foci and lymphoid depletion were present. Electron microscopy of the liver showed viral particles
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Fig. 2. Liver, turkey breeder hen, HE stain, fatty degeneration (right part), and transition (arrowheads) to necrotic focus with acute hemorrhages (left part).
of 23–28 nm, similar to parvovirus. No virus could be isolated, and nucleic acids of circovirus, polyomavirus, aviadenovirus, and parvovirus could not be detected by PCR. The toms kept on the same farm were not affected. Because intoxication via the feed was suspected, the feed was changed. Furthermore, birds were treated for 3 days with enrofloxacin (10 mg/kg bodyweight, Baytril 10%, Bayer Vital, Leverkusen, Germany), followed by treatment with amoxicillin (40 mg/kg body weight, Aciphen, Bela-Pharm GmbH und Co KG, Vechta, Germany) for 3 days. The antibiotics did not show any effect, but supplementation with a preparation containing 150 g of
vitamin E and 100 mg of selenium per liter (E + Selen 15%, Konivet, Essen, Germany) seemed to improve the clinical status. The preparation was given for 5 days at a dilution of 2:1000 in the drinking water. Additional cases. Between 2009 and 2011 similar cases were observed in seven other breeder flocks (Table 1) and in seven meat turkey flocks (Table 2). All cases in breeder flocks affected hens, while six of seven diseased meat turkey flocks were female. The age of the breeder hens was between 18 and 23 wk, while meat turkey flocks were aged between 12 and 16 wk. In breeder flocks the disease lasted between 4 days and 2 wk, and mortality was between 1.2%
Fig. 3. Liver, turkey breeder hen, HE stain, magnification of transition area with acute hemorrhages and multifocal eosinophilic, intranuclear inclusion bodies in hepatocytes (arrowheads).
2006
2009
2009
2010
2010
2010
2011
2011
1
2
3
4
5
6
7
8
3900
3900
2416
2244
5300
5450
8000
5200
Birds in flock
Hens
Hens
Hens
Hens
Hens
Hens
Hens
Hens
Sex
20
18
19
19
22–23
22
20
22–23
Age (wk)
Histological findings in the liver
Virological investigationA
CEF, Cir, Pol, AADV, Par: 5.31 in 14 days Severe dystrophy of the liver negative with necrosis, severe fatty degeneration of hepatocytes, eosinophilic intranuclear inclusions 1.37 in 5 days Multiple necrosis in the liver, CEF, Cir, Pol, AADV, Par: eosinophilic intranuclear negative inclusions CEF, Cir, Pol, AADV, Par: 0.66 in 6 days Severe fatty degeneration and negative necrotizing hepatitis with hemorrhages, intranuclear inclusions CEF, CEL, Cir, Pol, AADV, 2.83 in 7 days Severe fatty degeneration; Par: negative multifocal, acute, hemorrhagic, necrotizing hepatitis with eosinophilic intranuclear inclusions; subacute, lymphoplasmatic cholangitis CEF, Cir, Pol, AADV, Par, 1.2 in 4 days Fatty degeneration and mild chronic, interstitial lymphocytic Pic: negative; Aviadenovirus isolated from a pooled hepatitis sample of liver and heart on CEL; additionally a reovirus was isolated from a pooled sample of intestines and spleen and detected by RT-PCR 1.74 in 4 days Fatty degeneration and acute CEL, CEF, Cir, Pol, AADV, hemorrhages Par: negative 1.5 in 6 days Severe fatty degeneration of the CEF, Cir, Pol, AADV, Par, Reo, Pic: negative; no virus hepatocytes, mild hyperemia with degeneration of hepatocytes isolated from the liver on accumulation of bile pigment in CEL, but a reovirus isolated from the intestines hepatocytes; extramedullar on CEL hematopoesis 7.5 in 7 days Severe fatty degeneration CEL, CEF, Cir, Pol, AADV, Par: negative
Mortality (%)
Electron microscopy (liver)
Therapy
Three days after first losses vitamin E and selenium for 5 days
Immediately vitamin E and selenium Immediately vitamin E and selenium
Immediately vitamin E and selenium
Virus particles of 28–30 nm, Immediately vitamin E and similar to picornavirus selenium
Not done
Not done
Not done
Virus particles of 28–30 nm, Immediately vitamin E and similar to picornavirus selenium, initially in addition to aciphen/ amoxicillin, which was given due to respiratory disease
No virus particles found
Virus particles of 23–28 nm, Immediately enrofloxacin for similar to parvovirus 3 days, followed by amoxicillin for 3 days, followed by vitamin E and selenium Virus particles of 23–28 nm, Vitamin E and selenium similar to parvovirus
A If not indicated otherwise samples of the liver were investigated; virological investigations consisted of attempted virus isolation in chicken embryo liver cells (CEL) and chicken embryo fibroblasts (CEF) as well as PCR for detection of circovirus (Cir), polyomavirus (Pol), aviadenovirus (AADV), parvovirus (Par), reovirus (Reo), and picornavirus (Pic). Not all methods were tried in all cases.
Year
Diagnostic findings and therapy of eight cases with liver lesions and raised mortality in turkey breeder flocks.
Case
Table 1.
Liver changes in turkey flocks
477
2010
2010
2011
2011
2011
2011
2011
1
2
3
4
5
6
7
Hens
Hens
Sex
8500
12,500
6500
2000
Hens
Hens
Hens
Hens
5500 Toms
7200
4030
Birds in flock
13
16
16
13.5
14.5
12
14
Age (weeks)
0.9 in 1 day
0.5 in 3 days
,1.0 in 3 days
,1.0 in 2 days
,1.1 in 3 days
0.46 in 1 day
,1.0 in 3 days
Mortality %
Moderate to severe fatty degeneration
In some birds severe fatty degeneration, in other birds multifocal necrotizing hepatitis with intranuclear inclusion bodies suspected Moderate to severe hepatocellular necroses and/ or hepatocellular degeneration Severe hepatocellular degeneration with early centrolobular necrosis and hemorrhages; diffuse lymphoplasmatic cholangitis Severe hepatocellular degeneration with early centrolobular necrosis and hemorrhages; diffuse lymphoplasmatic cholangitis Hepatocellular necrosis with suspicion of intranuclear inclusion bodies suspected Severe dystrophic fatty degeneration
Histological findings in the liver
CEL, CEF, Cir, Pol, AADV, Par: negative CEL, Cir, Pol, AADV, Par: negative; in CEF a cytopathogenic effect was observed in the first, but not second passage CEF, Cir, Pol, AADV, Par: negative; an Fowl Adenovirus was isolated on CEL
CEL, CEF, Cir, Pol, AADV, Par: negative
CEF, CEL, Cir, Pol, AADV, Par, Reo: negative; Pic: positive CEL, CEF, Cir, Pol, AADV, Par: negative CEL, CEF, Cir, Pol, AADV, Par: negative
Virological investigationA
Diagnostic findings and therapy of eight cases with liver lesions and raised mortality in meat turkey flocks. Therapy
Immediately vitamin E and selenium Immediately vitamin E and selenium
Immediately vitamin E and selenium
Not done
Immediately vitamin E and selenium
Immediately vitamin E and selenium Immediately vitamin E and selenium
Immediately vitamin E and selenium
No virus particles found Not done
Not done
Not done
Not done
No virus particles found
Electron microscopy (liver)
A If not indicated otherwise samples of the liver were investigated; virological investigations consisted of attempted virus isolation in chicken embryo liver cells (CEL), chicken embryo fibroblasts (CEF), or chicken embryos (CE) as well as PCR for detection of circovirus (Cir), polyomavirus (Pol), aviadenovirus (AADV), parvovirus (Par), reovirus (Reo), and picornavirus (Pic). Not all methods were tried in all cases.
Year
Case
Table 2.
478 C. Popp et al.
479
Liver changes in turkey flocks
DISCUSSION
Fig. 4. Liver, turkey breeder hen, electron microscopy, virus particles similar to parvo- or picornavirus.
and 7.5%. In meat turkey flocks, the disease lasted only 1–3 days. with mortality around 1%. Postmortem findings were nearly identical to the first case described, but in some cases necroses in the pancreas were observed. Often E. coli were isolated from the livers. Twice C. perfringens was isolated from the liver; however isolation of C. perfringens was not tried in all cases. Histopathological findings were ambiguous. Fatty degeneration of hepatocytes with parenchymal collapse and associated hemorrhage were observed in all cases except one in breeder turkeys and three cases in meat turkeys (Fig. 2). Hepatitis or cholangitis was seen in four cases in breeders and three cases in meat turkeys. Intranuclear inclusion bodies in hepatocytes were observed in four cases in breeders, including the first case, and two cases in meat turkeys (Fig. 3). In three cases in breeders, virus particles, 23–30 nm in diameter and similar to parvo- or picornavirus, were detected by electron microscopy (Fig. 4). In one case in breeders and in two cases in meat turkeys, no virus particles were seen. In the other cases no electron microscopy was done. From the livers of one breeder flock and of one meat turkey flock an aviadenovirus was isolated. From the intestines and spleen of this breeder flock and from the intestines of another breeder flock a reovirus was isolated. Picornavirus RNA was detected in the livers of one meat turkey flock; otherwise, investigations by PCR yielded only negative results. In all cases, analysis of feed composition revealed no deviations (results not shown). In some cases it was observed that blood samples taken from turkeys during the acute phase of the disease did not coagulate. This was not observed with blood taken from turkeys showing no symptoms. Because treatment with vitamin E and selenium had improved the clinical signs in the first case, all further flocks showing these symptoms were treated with vitamin E and selenium, and the dosage of the preparation was doubled. In all cases symptoms disappeared within few days after the start of the treatment. Autogenous inactivated vaccines were produced as oil emulsions using the isolated aviadeno- and reoviruses (Vaxxinova, Cuxhaven, Germany). One dose contained 106 TCID50 of each virus. Since then, all breeder flocks on three different farms were vaccinated twice intramuscularly with this vaccine at the age of 10 or 11 and 16 wk. In vaccinated flocks only 5–11 hens died showing the described symptoms during another vaccination at the age of 25 wk.
The series of cases described here occurred between 2006 and 2011, and further similar cases have been observed after the last case included in this report. The disease was seen almost only in turkey hens. Breeder hens were aged between 18 and 23 wk, while meat turkey hens were aged between 12 and 16 wk. Clinical symptoms as well as pathological and histological findings were similar in all cases, while the results of bacteriological and virological examinations differed. The most prominent features of the disease were a suddenly increasing mortality and liver lesions. In most cases histopathological results showed a fatty degeneration of hepatocytes, lesion characteristic of lipidosis. Lipidosis occurs sporadically in turkeys, and especially in turkey hens, between 12 and 24 wk (13). However, there is only very little information available about the disease. In turkeys it was first described by Gazdzinski et al. (8). As in the cases described here, they observed a suddenly increasing mortality in 20-wk-old turkey breeder hens. Mortality was between 0.7% and 1.7% within 12 days. Affected birds were unable to walk and showed signs of dyspnea and cyanosis. Livers were enlarged with pale areas and numerous hemorrhages, and histology showed large vacuoles in the hepatocytes containing fat and massive hemorrhages with disrupted, degenerated, and necrotic tissue around them (8). Eight further cases were described by Manarolla et al. (19). While no information about clinical signs was given, histological lesions were similar to those described by Gazdzinski et al. (8). Four cases affected commercial turkey poults, both male and female, of about 1 wk of age. Two cases occurred in 4- and 12-wk-old birds, both of these flocks were female. Only twice were 20-wk-old birds affected: once for breeder hens and once for fattening toms (19). Thus age and sex of most affected flocks were different than in cases of lipidosis of turkeys as described in this report and others (8,13). A number of possible factors leading to lipidosis in turkeys have been suggested and discussed in detail by Hazel (13) and Gazdzinski et al. (8). Predisposing factors are mostly related to nutrition and include high energy consumption, especially in combination with low protein diets and particularly with diets low in the lipotropic factors methionine and cysteine, which are needed for the synthesis of apolipoproteins (13). Also diets deficient in carnitine, which is required for the transport of fatty acids from the cytosol into the mitochondria, predispose hepatic lipidosis in turkeys (13). All these factors may lead to an insufficient b-oxidation and the accumulation of fatty acids in the liver (4,13). Further, nonnutritional predisposing factors such as high environmental temperatures leading to an uneven feeding pattern during the day, high estrogen levels, toxic substances, and genetic defects are discussed, and some of them have been experimentally shown in layers with fatty liver syndrome (28). Stress caused by a change of the light program, a change of the feed, needle vaccination with handling of the birds, or previous diseases might have had an influence in some of the described cases. In addition to lipidosis, several microorganisms can cause liver lesions in turkeys that have to be considered for differential diagnosis. However, evidence for infectious causes was inconclusive. Histological lesions in the liver did not indicate bacterial involvement, even though E. coli was isolated from livers in a number of cases. This was interpreted as a postmortal or perimortal translocation of intestinal bacteria, rather than an etiological involvement, since lesions did not resemble lesions related to E. coli as described in the literature (3). While isolation of clostridia was not tried on a regular base, C. perfringens was isolated from affected livers in one case. Clostridia perfringens has been described as a
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causative agent of liver lesions and cholangiohepatitis in chickens (21,23) and turkeys (6), but lesions caused by C. perfringens are characterized by proliferation of bile ductules and cholecystitis, which were detected in only four cases described here. Indications for viral involvement were more ambiguous, however. In six cases eosinophilic intranuclear inclusion bodies were detected, indicative of a viral infection. On the other hand, eosinophilic nucleoli, which are prominent in the nuclei of degenerating hepatocytes, can be mistaken for viral inclusion bodies (4). In three of these cases small virus particles resembling picorna- or parvoviruses were detected by electron microscopy, and in another case picornavirus RNA was detected by PCR. While parvoviruses may cause intranuclear inclusion bodies, no parvovirus has been implicated with liver lesions in gallinaceous birds. On the other hand, recently a picornavirus was identified as the cause of turkey viral hepatitis (16). In spite of that turkey viral hepatitis seems an unlikely diagnosis, since it affects younger birds and histological lesions are characterized by necrosis of hepatocytes with granulocytic infiltration and hemorrhages, lacking the accumulation of lipids found here (12,26,27). Twice an aviadenovirus was isolated from affected livers. Aviadenoviruses can cause inclusion body hepatitis in broilers (1). Inclusion body hepatitis has been described in turkey poults between 1 day and 4 wk of age (9,10,25). Histological lesions were described as coagulative necrosis of hepatocytes with infiltration of inflammatory cells and with numerous basophilic intranuclear inclusion bodies in the hepatocyte (25). None of these signs were observed in the cases presented here. The lack of consistent microbiological findings raises the question of whether these cases are really related. It is possible, that cases were caused by different viral or bacterial pathogens, which were detected by the diagnostic procedures. However, it is unlikely that these different pathogens all affected birds of a similar age and caused similar lesions. Furthermore, it is possible that the lesions in some or all cases were caused by a hitherto unknown virus, which did not belong to the six virus families investigated by PCR and which did not grow in cell culture causing a cytopathological effect. Further investigations, including staining of cell cultures with indirect immunofluorescence using convalescent sera, would be necessary to detect that virus. Even though all signs, including the successful therapy with vitamin E and selenium, indicated a metabolic etiology, the disease did not reoccur in flocks that had been vaccinated with an autogenous vaccine containing the isolated aviadeno- and reovirus. Thus it is possible that viral infections contribute to the problem. In conclusion, the disease condition seems to be a multifactorial problem, in which hepatic lipidosis has a central role and to which different stressors, including viruses, contribute. REFERENCES 1. Adair, B. M., and S. D. Fitzgerald. Group I adenovirus infections. In: Diseases of poultry, 12th ed. Y. M. Saif, A. M. Fadly, J. R. Glisson, L. R. McDougald, L. K. Nolan, and D. E. Swayne, eds. Iowa State Press, Ames, IA. pp. 252–266. 2008. 2. Andersen, A. A., and D. Vanrompay. Avian chlamydiosis (psittacosis, ornithosis). In: Diseases of poultry, 12th ed. Y. M. Saif, A. M. Fadly, J. R. Glisson, L. R. McDougald, L. K. Nolan, and D. E. Swayne, eds. Iowa State Press, Ames, IA. pp. 863–882. 2008. 3. Barnes, H. J., L. K. Nolan, and J.-P. Vaillancourt. Colibacillosis. In: Diseases of poultry, 12th ed. Y. M. Saif, A. M. Fadly, J. R. Glisson, L. R. McDougald, L. K. Nolan, and D. E. Swayne, eds. Iowa State Press, Ames, IA. pp. 691–732. 2008.
4. Crespo, R., and H. L. Shivaprasad. Hepatic lipidosis of turkeys. In: Diseases of poultry, 12th ed. Y. M. Saif, A. M. Fadly, J. R. Glisson, L. R. McDougald, L. K. Nolan, and D. E. Swayne, eds. Iowa State Press, Ames, IA. pp. 1174–1175. 2008. 5. Doane, F. W., and N. Anderson. Electron microscopy in diagnostic virology. Press Syndicate of the University of Cambridge, New York. 1987. 6. Eleazer, T. H., and J. S. Harrell. Clostridium perfringens infection in turkey poults. Avian Dis. 20:774–776. 1976. 7. Fadly, A. L., and L. N. Payne. Leukosis/sarcoma group. In: Diseases of poultry, 12th ed. Y. M. Saif, A. M. Fadly, J. R. Glisson, L. R. McDougald, L. K. Nolan, and D. E. Swayne, eds. Iowa State Press, Ames, IA. pp. 407–465. 2008. 8. Gazdzinski, P., E. J. Squires, and R. J. Julian. Hepatic lipidosis in turkeys. Avian Dis. 38:379–384. 1994. 9. Guy, J. S., and H. J. Barnes. Characterization of an avian adenovirus associated with inclusion body hepatitis in day-old turkeys. Avian Dis. 41:726–731. 1997. 10. Guy, J. S., J. L. Schaeffer, and H. J. Barnes. Inclusion-body hepatitis in day-old turkeys. Avian Dis. 32:587–590. 1988. 11. Halami, M. Y., H. Nieper, H. Mu¨ller, and R. Johne. Detection of a novel circovirus in mute swans (Cygnus olor) by using nested broad-spectrum PCR. Virus Res. 132:208–212. 2008. 12. Hauck, R., R. P. Chin, G. Sentı´es-Cue´, B. Charlton, and H. L. Shivaprasad. Retrospective study of turkey viral hepatitis in California turkey flocks, 2000–2012. Avian Dis. 58:205–210. 2014. 13. Hazel, K. Hepatic lipidosis: is carnitine deficiency the underlaying cause? In: Proceedings of the 5th International Meeting of the Working Group 10. H. M. Hafez, ed. Mensch & Buch Verlag, Berlin. pp. 271–277. 2009. 14. Hazelton, P. R., and H. R. Gelderblom. Electron microscopy for rapid diagnosis of emerging infectious agents. Emerg. Infect. Dis. 9:294–303. 2003. 15. Hoerr, F. J. Mycotoxicoses. In: Diseases of poultry, 12th ed. Y. M. Saif, A. M. Fadly, J. R. Glisson, L. R. McDougald, L. K. Nolan, and D. E. Swayne, eds. Iowa State Press, Ames, IA. pp. 1103–1133. 2008. 16. Honkavuori, K. S., H. L. Shivaprasad, T. Briese, C. Street, D. L. Hirschberg, S. K. Hutchison, and W. I. Lipkin. Novel picornavirus in turkey poults with hepatitis, California, USA. Emerg. Infect. Dis. 17:480–487. 2011. 17. Johne, R., D. Enderlein, H. Nieper, and H. Mu¨ller. Novel polyomavirus detected in the feces of a chimpanzee by nested broadspectrum PCR. J. Virol. 79:3883–3887. 2005. 18. Madeley, C. R., and A. M. Field. Virus morphology. Churchill Livingstone, Philadelphia, PA. 1988. 19. Manarolla, G., M. Saita, D. Gallazzi, G. Sironi, and T. Rampin. Histological and histochemical investigations on hepatic lipidosis in turkeys. Arch. Geflu¨gelkd. 75:56–60. 2011. 20. McDougald, L. R. Histomoniasis (Blackhead) and other protozoan diseases of the intestinal tract. In: Diseases of poultry, 12th ed. Y. M. Saif, A. M. Fadly, J. R. Glisson, L. R. McDougald, L. K. Nolan, and D. E. Swayne, eds. Iowa State Press, Ames, IA. pp. 1095–1105. 2008. 21. Onderka, D. K., C. C. Langevin, and J. A. Hanson. Fibrosing cholehepatitis in broiler chickens induced by bile duct ligations or inoculation of Clostridium perfringens. Can. J. Vet. Res. 54:285–290. 1990. 22. Raue, R., and M. Hess. Hexon based PCRs combined with restriction enzyme analysis for rapid detection and differentiation of fowl adenoviruses and egg drop syndrome virus. J. Virol. Methods 73:211–217. 1998. 23. Sasaki, J., M. Goryo, and K. Okada. Cholangiohepatitis in chickens induced by bile duct ligations and inoculation of Clostridium perfringens. Avian Pathol. 29:405–410. 2000. 24. Schat, K. A., and H. G. Purchase. Cell-culture methods. In: A laboratory manual for the isolation and identification of avian pathogens, 3rd ed. H. G. Purchase, L. H. Arp, C. H. Domermuth, and J. E. Pearson, eds. Kendall/Hunt Publishing Company, Dubuque, IA. pp. 167–175. 1989. 25. Shivaprasad, H. L., P. R. Woolcock, and M. D. McFarland. Group I avian adenovirus and avian adeno-associated virus in turkey poults with inclusion body hepatitis. Avian Pathol. 30:661–666. 2001.
Liver changes in turkey flocks
26. Snoeyenbos, G. H., and H. I. Basch. Further studies of virus hepatitis of turkeys. Avian Dis. 4:477. 1960. 27. Snoeyenbos, G. H., H. I. Basch, and M. Sevoian. An infectious agent producing hepatitis in turkeys. Avian Dis. 3:377. 1959. 28. Squires, E. J., and S. Leeson. Aetiology of fatty liver syndrome in laying hens. Br. Vet. J. 144:602–609. 1988. 29. Wellehan, J. F. X. Jr., A. L. Childress, R. E. Marschang, A. J. Johnson, E. W. Lamirande, J. F. Roberts, M. L. Vickers, J. M. Gaskin, and E. R. Jacobson. Consensus nested PCR amplification and sequencing of diverse reptilian, avian, and mammalian orthoreoviruses. Vet. Microbiol. 133:34–42. 2009.
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30. Witter, R. L., and K. A. Schat. Marek’s disease. In: Diseases of poultry, 12th ed. Y. M. Saif, A. M. Fadly, J. R. Glisson, L. R. McDougald, L. K. Nolan, and D. E. Swayne, eds. Iowa State Press, Ames, IA. pp. 407–465. 2008. 31. Xie, Z., M. I. Khan, T. Girshick, and Z. Xie. Reverse transcriptasepolymerase chain reaction to detect avian encephalomyelitis virus. Avian Dis. 49:227–230. 2005. 32. Zsak, L., K. O. Strother, and J. M. Day. Development of a polymerase chain reaction procedure for detection of chicken and turkey parvoviruses. Avian Dis. 53:83–88. 2009.
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AVIAN DISEASES 58:474–481, 2014
Case Report— Liver Pathology Associated with Increased Mortality in Turkey Breeder and Meat Turkey Flocks Christina Popp,A Ru¨diger Hauck,BF Thomas W. Vahlenkamp,C Do¨rte Lu¨schow,B Olivia Kershaw,D Marc Hoferer,E and Hafez M. HafezBG A Poultry Health Service Baden-Wu¨rttemberg, Schaflandstr. 3/3, 70736 Fellbach, Germany Institute for Poultry Diseases, Freie Universita¨t Berlin, Ko¨nigsweg 63, 14163 Berlin, Germany C Institute of Virology, University of Leipzig, An den Tierkliniken 29, 04103 Leipzig, Germany D Department of Veterinary Pathology, Freie Universita¨t Berlin, Robert-von-Ostertag-Strasse 15, 14163 Berlin, Germany E Chemical and Veterinary Investigations Office, Stuttgart (CVUAS), Schaflandstr. 3/3, 70736 Fellbach, Germany B
Received 1 October 2013; Accepted 17 February 2014; Published ahead of print 17 February 2014 SUMMARY. Between 2006 and 2011 a series of disease conditions characterized by raised mortality and liver disorders occurred in turkey breeder flocks and in meat turkey flocks in Germany. The flocks were between 12 and 23 wk of age, and mostly hens were affected. Clinical signs were nonspecific and accompanied by mortality varying between 1% and 7%. Affected birds displayed swollen livers that were marbled with black and red spots and yellowish areas. The pericardium was filled with an amber fluid, and the coronary groove was extensively filled with fat. Spleens were swollen, and a serous fluid that seemed to leak from the liver was present in the body cavity. Histopathological findings in all but one case included fatty degeneration of hepatocytes with parenchymal collapse and associated hemorrhages. Some animals showed cholangitis and hepatitis with intranuclear inclusion bodies. In three cases with breeders, electron microscopy detected virus particles that were between 23 and 30 nm and similar to parvo- or picornavirus. In addition, picornavirus RNA was detected in the livers of one meat turkey flock. Investigations by PCR for circovirus, polyomavirus parvovirus, and aviadenovirus yielded negative results in all cases, but an aviadenovirus was isolated from livers twice and a reovirus from the intestines once. Supplementation with vitamin E and selenium seemed to improve the situation. The most likely diagnosis is lipidosis, a metabolic disorder with complex etiology, which has rarely been described in turkeys. RESUMEN. Reporte de Caso—Patologı´a hepa´tica asociada con aumento de mortalidad en parvadas de pavos reproductores y de engorde. Entre 2006 y 2011 se presentaron una serie de enfermedades caracterizadas por aumento de la mortalidad y problemas hepa´ticos en parvadas de pavos reproductores y de engorde en Alemania. Las parvadas tenı´an entre 12 y 23 semanas de edad y en su mayorı´a las gallinas fueron las ma´s afectadas. Los signos clı´nicos fueron inespecı´ficos y estuvieron acompan˜ados por mortalidad que oscilo´ entre el 1% y 7%. Las aves afectadas mostraron hı´gados inflamados que presentaban aspecto marmoleado con a´reas amarillentas y puntos negros y rojos. El pericardio estaba lleno de un fluido de color a´mbar, y el surco coronario estaba extensivamente lleno de grasa. Los bazos se observaron inflamados y un fluido seroso que parecı´a salir del hı´gado, estaba presente en la cavidad corporal. Los hallazgos histopatolo´gicos en todos los casos menos en uno incluyen degeneracio´n grasa de los hepatocitos con colapso del pare´nquima y hemorragias asociadas. Algunos animales mostraron colangitis y hepatitis con cuerpos de inclusio´n intranucleares. En tres casos con reproductores, la microscopı´a electro´nica detecto´ partı´culas virales que oscilaban entre 23 y 30 nm, similares a parvo o picornavirus. Adema´s, se detecto´ ARN de picornavirus en los hı´gados de una parvada de pavos de engorde. Las investigaciones por PCR para circovirus, parvovirus, poliomavirus y aviadenovirus mostraron resultados negativos en todos los casos, pero se aislo´ un aviadenovirus a partir de hı´gados dos veces y un reovirus de los intestinos en una vez. La suplementacio´n con vitamina E y el selenio parecieron mejorar la situacio´n. El diagno´stico ma´s probable fue lipidosis, un trastorno metabo´lico con etiologı´a compleja, que rara vez se ha descrito en pavos. Key words: turkeys, metabolic disorders, lipidosis Abbreviations: CE 5 chicken embryo; DMEM 5 Dulbecco’s modified Eagle’s medium; HE 5 hematoxylin and eosin; PBS 5 phosphate-buffered saline; RT 5 reverse transcription
The liver is the main metabolic organ of birds. Among its tasks are detoxication and formation of blood proteins. It is also an important site of lipid metabolism. Diseases of the liver in poultry can be caused by infectious or noninfectious causes. Among the numerous pathogens causing liver lesions are viruses (aviadenoviruses (1), picornavirus (16), tumor viruses (7,30)), bacteria (Escherichia coli F Present address: Federal Office for Consumer Protection and Food Safety, Mauerstr. 39-42, 10117 Berlin, Germany G Corresponding author. E-mail: [email protected]
(3), Clostridium perfringens (6,21,23), Chlamydia psittaci (2)), and the protozoan parasite Histomonas meleagridis (20). Noninfectious causes include intoxications, especially with mycotoxins (15), and metabolic disorders like hepatic lipidosis (4,8). During the last several years a series of disease conditions characterized by liver disorders and accompanied by increased mortality occurred in turkey breeder flocks and in meat turkey flocks in Germany. Here we describe several cases that occurred in the state of Baden-Wu¨rttemberg in Southern Germany between 2006 and 2011.
474
475
Liver changes in turkey flocks
Fig. 1.
Typical liver appearance in diseased birds in two cases in breeder hens (A) and meat turkey hens (B).
MATERIALS AND METHODS Microbiology. Samples from heart blood, livers, and lungs were collected with sterile disposable bacterial loops and streaked on Columbia agar containing 7% sheep blood and Gassner agar (Oxoid, Wesel, Germany). Plates were incubated at 37uC overnight under aerobic conditions. Histopathology. Liver samples were fixed in 4% formalin and embedded in paraffin following standard laboratory procedures. Sections (3 mm) of dewaxed tissues were stained with hematoxylin and eosin (HE) for histopathological investigation. Electron microscopy. After negative staining, transmission electron microscopy (80 keV microscope JEM-1011; Jeol, Eching, Germany) was used to identify viral particles in livers. One grid per sample was prepared. Briefly, after the grids were cleaned in an ultrasonic bath using acetic acid and washed, they were coated with a 0.8% solution of pioloform (PLANO, Wetzlar, Germany) and ROTIPURANH chloroform (Roth, Karlsruhe, Germany). After cell disruption of the tissue samples (50 mg of tissue in 500 ml of double-distilled water) with a TeSeE PRECESSH 24 homogenizer (Bio-Rad, Munich, Germany), the samples were centrifuged at 420 3 g for 10 min, and the supernatants transferred onto a Parafilm ‘‘M’’ (Brand, Wertheim, Germany). Subsequently, the glow discharge-pretreated carbon grids were coated with the different samples. Phosphotungstic acid solution (1% [w/v], pH 7.2) was used for negative staining. Images (200,0003 amplification) were taken with the SIS Megaview III CCD Olympus camera. The software AnalySIS (Olympus, Mu¨nster, Germany) was used to take size measurements of the viruses and to identify typical structural criteria mentioned in the literature (5,14,18). Virus isolation. Chicken embryo (CE) fibroblasts and CE liver cells were prepared from 10-day-old embryos according to standard protocols (24). Cells were grown as monolayer until near confluence was obtained. Primary and secondary cultures of CE cells were maintained at 38 C and 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% fetal calf serum, 50 U/ml penicillin, and 50 mg/ml streptomycin. For infection experiments, CE cells were washed with phosphate-buffered saline (PBS) and incubated with organ supernatants. These were obtained by mincing 1 g of organ material with sand and 10 ml of PBS containing 100 U/ml penicillin and 100 mg/ml streptomycin in a mortar under sterile conditions and subsequent incubation for 30 min at room temperature. After two freeze–thaw cycles at 280 C the virus containing supernatant was harvested after centrifugation at 1500 3 g for 5 min. The supernatant was filter sterilized with a 0.20-mm filter, and 500 ml was used for each well of a 24-well plate. All infections were performed in duplicate. Mock-infected cells were used as controls. After incubation with the supernatants for 60 min, CE cells were rinsed and DMEM was added as already described. Supernatants were harvested when a cytopathic effect had developed, usually at 3 days after infection. Polymerase chain reaction. The isolation of nucleic acids from organ samples was performed by mincing 25 mg of the organ material with sand under sterile conditions following centrifugation at room temperature at
1048 3 g for 5 min. The supernatants of the organ suspensions were subjected to DNA or RNA isolation using the DNeasy and RNeasy Mini Kit (Qiagen, Hilden, Germany), respectively. Five microliters of the purified nucleic acids was used for the subsequent amplification. The presence of avian circoviruses was analyzed according to Halami et al. (11) using a consensus nested PCR, which enables the detection of a broad spectrum of avian circoviruses. The nested PCR amplified a product of 350 bp in length. Polyomavirus was detected according to Johne et al. (17) using a consensus nested PCR, which enables the detection of a broad spectrum of polyomaviruses in different species. The nested PCR amplified a 273bp product. The presence of aviadenovirus was analyzed using PCR, which amplified a product of 590 bp (22). Parvovirus was analyzed according to Zsak et al. (32). The PCR generated a product of 561 bp in length. Avian reovirus was detected according to Wellehan et al. (29) using a consensus reverse transcription (RT) nested PCR targeting conserved regions of the viral polymerase gene. The RT-PCR generated a 226 bpproduct. Avian picornavirus was detected according to Xie et al. (31) using primers targeting the VP2 gene of avian encephalomyelitis virus. The RT step generated a 619-bp cDNA product, which was subsequently amplified.
CASE REPORT
The initial case. The disease was first observed in 2006 in a flock of 5250 turkey breeder hens. At the age of 22 to 23 wk mortality started to rise. Initially about four to six birds died each day, but the number of daily fatalities increased up to 80 birds a day. Within 2 wk, 5.3% of the birds died. Affected birds withdrew from the other birds, laid down, closed their eyes, dropped their heads, flapped their wings, and gasped. Furthermore, they bled out of the beak. Birds died within 2–6 hr after showing these symptoms. At postmortem, livers were swollen, marbled with black and red spots, and showed yellow areas (Fig. 1). The pericardium was filled with an amber fluid, the coronary groove was extensively filled with fat, and spleens were swollen. A blood-tinged fluid was in the abdominal cavity due to liver rupture. Furthermore, catarrhal enteritis was observed. Escherichia coli was isolated from the hearts, livers, and lungs of some birds. Histopathological investigation identified a severe hepatic dystrophy with severe fatty degeneration and centrilobular necroses, disseminated hemorrhages, and periportal infiltrations (Fig. 2). Eosinophilic intranuclear inclusion bodies suggested a viral infection (Fig. 3). Centrifollicular and perivascular fibrinous exudates admixed with extracellular and intrahistiocytic bacteria were present in the spleen. Furthermore, a few necrotic foci and lymphoid depletion were present. Electron microscopy of the liver showed viral particles
476
C. Popp et al.
Fig. 2. Liver, turkey breeder hen, HE stain, fatty degeneration (right part), and transition (arrowheads) to necrotic focus with acute hemorrhages (left part).
of 23–28 nm, similar to parvovirus. No virus could be isolated, and nucleic acids of circovirus, polyomavirus, aviadenovirus, and parvovirus could not be detected by PCR. The toms kept on the same farm were not affected. Because intoxication via the feed was suspected, the feed was changed. Furthermore, birds were treated for 3 days with enrofloxacin (10 mg/kg bodyweight, Baytril 10%, Bayer Vital, Leverkusen, Germany), followed by treatment with amoxicillin (40 mg/kg body weight, Aciphen, Bela-Pharm GmbH und Co KG, Vechta, Germany) for 3 days. The antibiotics did not show any effect, but supplementation with a preparation containing 150 g of
vitamin E and 100 mg of selenium per liter (E + Selen 15%, Konivet, Essen, Germany) seemed to improve the clinical status. The preparation was given for 5 days at a dilution of 2:1000 in the drinking water. Additional cases. Between 2009 and 2011 similar cases were observed in seven other breeder flocks (Table 1) and in seven meat turkey flocks (Table 2). All cases in breeder flocks affected hens, while six of seven diseased meat turkey flocks were female. The age of the breeder hens was between 18 and 23 wk, while meat turkey flocks were aged between 12 and 16 wk. In breeder flocks the disease lasted between 4 days and 2 wk, and mortality was between 1.2%
Fig. 3. Liver, turkey breeder hen, HE stain, magnification of transition area with acute hemorrhages and multifocal eosinophilic, intranuclear inclusion bodies in hepatocytes (arrowheads).
2006
2009
2009
2010
2010
2010
2011
2011
1
2
3
4
5
6
7
8
3900
3900
2416
2244
5300
5450
8000
5200
Birds in flock
Hens
Hens
Hens
Hens
Hens
Hens
Hens
Hens
Sex
20
18
19
19
22–23
22
20
22–23
Age (wk)
Histological findings in the liver
Virological investigationA
CEF, Cir, Pol, AADV, Par: 5.31 in 14 days Severe dystrophy of the liver negative with necrosis, severe fatty degeneration of hepatocytes, eosinophilic intranuclear inclusions 1.37 in 5 days Multiple necrosis in the liver, CEF, Cir, Pol, AADV, Par: eosinophilic intranuclear negative inclusions CEF, Cir, Pol, AADV, Par: 0.66 in 6 days Severe fatty degeneration and negative necrotizing hepatitis with hemorrhages, intranuclear inclusions CEF, CEL, Cir, Pol, AADV, 2.83 in 7 days Severe fatty degeneration; Par: negative multifocal, acute, hemorrhagic, necrotizing hepatitis with eosinophilic intranuclear inclusions; subacute, lymphoplasmatic cholangitis CEF, Cir, Pol, AADV, Par, 1.2 in 4 days Fatty degeneration and mild chronic, interstitial lymphocytic Pic: negative; Aviadenovirus isolated from a pooled hepatitis sample of liver and heart on CEL; additionally a reovirus was isolated from a pooled sample of intestines and spleen and detected by RT-PCR 1.74 in 4 days Fatty degeneration and acute CEL, CEF, Cir, Pol, AADV, hemorrhages Par: negative 1.5 in 6 days Severe fatty degeneration of the CEF, Cir, Pol, AADV, Par, Reo, Pic: negative; no virus hepatocytes, mild hyperemia with degeneration of hepatocytes isolated from the liver on accumulation of bile pigment in CEL, but a reovirus isolated from the intestines hepatocytes; extramedullar on CEL hematopoesis 7.5 in 7 days Severe fatty degeneration CEL, CEF, Cir, Pol, AADV, Par: negative
Mortality (%)
Electron microscopy (liver)
Therapy
Three days after first losses vitamin E and selenium for 5 days
Immediately vitamin E and selenium Immediately vitamin E and selenium
Immediately vitamin E and selenium
Virus particles of 28–30 nm, Immediately vitamin E and similar to picornavirus selenium
Not done
Not done
Not done
Virus particles of 28–30 nm, Immediately vitamin E and similar to picornavirus selenium, initially in addition to aciphen/ amoxicillin, which was given due to respiratory disease
No virus particles found
Virus particles of 23–28 nm, Immediately enrofloxacin for similar to parvovirus 3 days, followed by amoxicillin for 3 days, followed by vitamin E and selenium Virus particles of 23–28 nm, Vitamin E and selenium similar to parvovirus
A If not indicated otherwise samples of the liver were investigated; virological investigations consisted of attempted virus isolation in chicken embryo liver cells (CEL) and chicken embryo fibroblasts (CEF) as well as PCR for detection of circovirus (Cir), polyomavirus (Pol), aviadenovirus (AADV), parvovirus (Par), reovirus (Reo), and picornavirus (Pic). Not all methods were tried in all cases.
Year
Diagnostic findings and therapy of eight cases with liver lesions and raised mortality in turkey breeder flocks.
Case
Table 1.
Liver changes in turkey flocks
477
2010
2010
2011
2011
2011
2011
2011
1
2
3
4
5
6
7
Hens
Hens
Sex
8500
12,500
6500
2000
Hens
Hens
Hens
Hens
5500 Toms
7200
4030
Birds in flock
13
16
16
13.5
14.5
12
14
Age (weeks)
0.9 in 1 day
0.5 in 3 days
,1.0 in 3 days
,1.0 in 2 days
,1.1 in 3 days
0.46 in 1 day
,1.0 in 3 days
Mortality %
Moderate to severe fatty degeneration
In some birds severe fatty degeneration, in other birds multifocal necrotizing hepatitis with intranuclear inclusion bodies suspected Moderate to severe hepatocellular necroses and/ or hepatocellular degeneration Severe hepatocellular degeneration with early centrolobular necrosis and hemorrhages; diffuse lymphoplasmatic cholangitis Severe hepatocellular degeneration with early centrolobular necrosis and hemorrhages; diffuse lymphoplasmatic cholangitis Hepatocellular necrosis with suspicion of intranuclear inclusion bodies suspected Severe dystrophic fatty degeneration
Histological findings in the liver
CEL, CEF, Cir, Pol, AADV, Par: negative CEL, Cir, Pol, AADV, Par: negative; in CEF a cytopathogenic effect was observed in the first, but not second passage CEF, Cir, Pol, AADV, Par: negative; an Fowl Adenovirus was isolated on CEL
CEL, CEF, Cir, Pol, AADV, Par: negative
CEF, CEL, Cir, Pol, AADV, Par, Reo: negative; Pic: positive CEL, CEF, Cir, Pol, AADV, Par: negative CEL, CEF, Cir, Pol, AADV, Par: negative
Virological investigationA
Diagnostic findings and therapy of eight cases with liver lesions and raised mortality in meat turkey flocks. Therapy
Immediately vitamin E and selenium Immediately vitamin E and selenium
Immediately vitamin E and selenium
Not done
Immediately vitamin E and selenium
Immediately vitamin E and selenium Immediately vitamin E and selenium
Immediately vitamin E and selenium
No virus particles found Not done
Not done
Not done
Not done
No virus particles found
Electron microscopy (liver)
A If not indicated otherwise samples of the liver were investigated; virological investigations consisted of attempted virus isolation in chicken embryo liver cells (CEL), chicken embryo fibroblasts (CEF), or chicken embryos (CE) as well as PCR for detection of circovirus (Cir), polyomavirus (Pol), aviadenovirus (AADV), parvovirus (Par), reovirus (Reo), and picornavirus (Pic). Not all methods were tried in all cases.
Year
Case
Table 2.
478 C. Popp et al.
479
Liver changes in turkey flocks
DISCUSSION
Fig. 4. Liver, turkey breeder hen, electron microscopy, virus particles similar to parvo- or picornavirus.
and 7.5%. In meat turkey flocks, the disease lasted only 1–3 days. with mortality around 1%. Postmortem findings were nearly identical to the first case described, but in some cases necroses in the pancreas were observed. Often E. coli were isolated from the livers. Twice C. perfringens was isolated from the liver; however isolation of C. perfringens was not tried in all cases. Histopathological findings were ambiguous. Fatty degeneration of hepatocytes with parenchymal collapse and associated hemorrhage were observed in all cases except one in breeder turkeys and three cases in meat turkeys (Fig. 2). Hepatitis or cholangitis was seen in four cases in breeders and three cases in meat turkeys. Intranuclear inclusion bodies in hepatocytes were observed in four cases in breeders, including the first case, and two cases in meat turkeys (Fig. 3). In three cases in breeders, virus particles, 23–30 nm in diameter and similar to parvo- or picornavirus, were detected by electron microscopy (Fig. 4). In one case in breeders and in two cases in meat turkeys, no virus particles were seen. In the other cases no electron microscopy was done. From the livers of one breeder flock and of one meat turkey flock an aviadenovirus was isolated. From the intestines and spleen of this breeder flock and from the intestines of another breeder flock a reovirus was isolated. Picornavirus RNA was detected in the livers of one meat turkey flock; otherwise, investigations by PCR yielded only negative results. In all cases, analysis of feed composition revealed no deviations (results not shown). In some cases it was observed that blood samples taken from turkeys during the acute phase of the disease did not coagulate. This was not observed with blood taken from turkeys showing no symptoms. Because treatment with vitamin E and selenium had improved the clinical signs in the first case, all further flocks showing these symptoms were treated with vitamin E and selenium, and the dosage of the preparation was doubled. In all cases symptoms disappeared within few days after the start of the treatment. Autogenous inactivated vaccines were produced as oil emulsions using the isolated aviadeno- and reoviruses (Vaxxinova, Cuxhaven, Germany). One dose contained 106 TCID50 of each virus. Since then, all breeder flocks on three different farms were vaccinated twice intramuscularly with this vaccine at the age of 10 or 11 and 16 wk. In vaccinated flocks only 5–11 hens died showing the described symptoms during another vaccination at the age of 25 wk.
The series of cases described here occurred between 2006 and 2011, and further similar cases have been observed after the last case included in this report. The disease was seen almost only in turkey hens. Breeder hens were aged between 18 and 23 wk, while meat turkey hens were aged between 12 and 16 wk. Clinical symptoms as well as pathological and histological findings were similar in all cases, while the results of bacteriological and virological examinations differed. The most prominent features of the disease were a suddenly increasing mortality and liver lesions. In most cases histopathological results showed a fatty degeneration of hepatocytes, lesion characteristic of lipidosis. Lipidosis occurs sporadically in turkeys, and especially in turkey hens, between 12 and 24 wk (13). However, there is only very little information available about the disease. In turkeys it was first described by Gazdzinski et al. (8). As in the cases described here, they observed a suddenly increasing mortality in 20-wk-old turkey breeder hens. Mortality was between 0.7% and 1.7% within 12 days. Affected birds were unable to walk and showed signs of dyspnea and cyanosis. Livers were enlarged with pale areas and numerous hemorrhages, and histology showed large vacuoles in the hepatocytes containing fat and massive hemorrhages with disrupted, degenerated, and necrotic tissue around them (8). Eight further cases were described by Manarolla et al. (19). While no information about clinical signs was given, histological lesions were similar to those described by Gazdzinski et al. (8). Four cases affected commercial turkey poults, both male and female, of about 1 wk of age. Two cases occurred in 4- and 12-wk-old birds, both of these flocks were female. Only twice were 20-wk-old birds affected: once for breeder hens and once for fattening toms (19). Thus age and sex of most affected flocks were different than in cases of lipidosis of turkeys as described in this report and others (8,13). A number of possible factors leading to lipidosis in turkeys have been suggested and discussed in detail by Hazel (13) and Gazdzinski et al. (8). Predisposing factors are mostly related to nutrition and include high energy consumption, especially in combination with low protein diets and particularly with diets low in the lipotropic factors methionine and cysteine, which are needed for the synthesis of apolipoproteins (13). Also diets deficient in carnitine, which is required for the transport of fatty acids from the cytosol into the mitochondria, predispose hepatic lipidosis in turkeys (13). All these factors may lead to an insufficient b-oxidation and the accumulation of fatty acids in the liver (4,13). Further, nonnutritional predisposing factors such as high environmental temperatures leading to an uneven feeding pattern during the day, high estrogen levels, toxic substances, and genetic defects are discussed, and some of them have been experimentally shown in layers with fatty liver syndrome (28). Stress caused by a change of the light program, a change of the feed, needle vaccination with handling of the birds, or previous diseases might have had an influence in some of the described cases. In addition to lipidosis, several microorganisms can cause liver lesions in turkeys that have to be considered for differential diagnosis. However, evidence for infectious causes was inconclusive. Histological lesions in the liver did not indicate bacterial involvement, even though E. coli was isolated from livers in a number of cases. This was interpreted as a postmortal or perimortal translocation of intestinal bacteria, rather than an etiological involvement, since lesions did not resemble lesions related to E. coli as described in the literature (3). While isolation of clostridia was not tried on a regular base, C. perfringens was isolated from affected livers in one case. Clostridia perfringens has been described as a
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causative agent of liver lesions and cholangiohepatitis in chickens (21,23) and turkeys (6), but lesions caused by C. perfringens are characterized by proliferation of bile ductules and cholecystitis, which were detected in only four cases described here. Indications for viral involvement were more ambiguous, however. In six cases eosinophilic intranuclear inclusion bodies were detected, indicative of a viral infection. On the other hand, eosinophilic nucleoli, which are prominent in the nuclei of degenerating hepatocytes, can be mistaken for viral inclusion bodies (4). In three of these cases small virus particles resembling picorna- or parvoviruses were detected by electron microscopy, and in another case picornavirus RNA was detected by PCR. While parvoviruses may cause intranuclear inclusion bodies, no parvovirus has been implicated with liver lesions in gallinaceous birds. On the other hand, recently a picornavirus was identified as the cause of turkey viral hepatitis (16). In spite of that turkey viral hepatitis seems an unlikely diagnosis, since it affects younger birds and histological lesions are characterized by necrosis of hepatocytes with granulocytic infiltration and hemorrhages, lacking the accumulation of lipids found here (12,26,27). Twice an aviadenovirus was isolated from affected livers. Aviadenoviruses can cause inclusion body hepatitis in broilers (1). Inclusion body hepatitis has been described in turkey poults between 1 day and 4 wk of age (9,10,25). Histological lesions were described as coagulative necrosis of hepatocytes with infiltration of inflammatory cells and with numerous basophilic intranuclear inclusion bodies in the hepatocyte (25). None of these signs were observed in the cases presented here. The lack of consistent microbiological findings raises the question of whether these cases are really related. It is possible, that cases were caused by different viral or bacterial pathogens, which were detected by the diagnostic procedures. However, it is unlikely that these different pathogens all affected birds of a similar age and caused similar lesions. Furthermore, it is possible that the lesions in some or all cases were caused by a hitherto unknown virus, which did not belong to the six virus families investigated by PCR and which did not grow in cell culture causing a cytopathological effect. Further investigations, including staining of cell cultures with indirect immunofluorescence using convalescent sera, would be necessary to detect that virus. Even though all signs, including the successful therapy with vitamin E and selenium, indicated a metabolic etiology, the disease did not reoccur in flocks that had been vaccinated with an autogenous vaccine containing the isolated aviadeno- and reovirus. Thus it is possible that viral infections contribute to the problem. In conclusion, the disease condition seems to be a multifactorial problem, in which hepatic lipidosis has a central role and to which different stressors, including viruses, contribute. REFERENCES 1. Adair, B. M., and S. D. Fitzgerald. Group I adenovirus infections. In: Diseases of poultry, 12th ed. Y. M. Saif, A. M. Fadly, J. R. Glisson, L. R. McDougald, L. K. Nolan, and D. E. Swayne, eds. Iowa State Press, Ames, IA. pp. 252–266. 2008. 2. Andersen, A. A., and D. Vanrompay. Avian chlamydiosis (psittacosis, ornithosis). In: Diseases of poultry, 12th ed. Y. M. Saif, A. M. Fadly, J. R. Glisson, L. R. McDougald, L. K. Nolan, and D. E. Swayne, eds. Iowa State Press, Ames, IA. pp. 863–882. 2008. 3. Barnes, H. J., L. K. Nolan, and J.-P. Vaillancourt. Colibacillosis. In: Diseases of poultry, 12th ed. Y. M. Saif, A. M. Fadly, J. R. Glisson, L. R. McDougald, L. K. Nolan, and D. E. Swayne, eds. Iowa State Press, Ames, IA. pp. 691–732. 2008.
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