Epidemiological and pathological investigation of fowl aviadenovirus serotypes 8b and 11 isolated from chickens with inclusion body hepatitis in Spain (2011–2013)

Inclusion body hepatitis caused by different fowl aviadenovirus (FAdV) serotypes has been described in several countries in recent years. In Spain, from the spring of 2011 to 2013, an increased number of outbreaks in broiler and broiler breeder flocks from different regions occurred. The objectives of the present work were to carry out the molecular characterization of FAdV strains from Spanish inclusion body hepatitis cases and to study the pathogenicity and viral dynamics of these strains in specific pathogen-free (SPF) chickens. A total of 52 inclusion body hepatitis clinical cases, including 45 from broiler farms and seven from broiler breeder farms, were analysed by conventional polymerase chain reaction and sequencing targeting the FAdV hexon gene. From these, 37 strains were classified as FAdV type 8b, while the remaining 15 were classified as FAdV types 11 (n?=?10), 2 (n?=?4) and 8a (n?=?1). In addition, two different FAdVs belonging to the genotypes 8b and 11 were used for experimental infection. Specific pathogen-free five-day-old birds were inoculated intramuscularly with a high (10^6.5 tissue culture infective dose (TCID)₅₀/ml) or low (10⁴ TCID₅₀/ml) dose of the above-mentioned FAdVs. No mortality was observed in any of the experimental groups, and only one bird showed evident clinical signs. However, macroscopic and microscopic hepatic lesions, as well as viral DNA, were detected in birds from all infection groups. Inclusion bodies and viral DNA were also detected in the pancreas and in the small and the large intestine in some birds. Long-lasting shedding and transmission to contact birds were confirmed in all infected groups.     

Evaluation of tiamulin and lincomycin for the treatment of broiler breeders experimentally infected with the intestinal spirochaete Brachyspira pilosicoli

Brachyspira pilosicoli strain CPSp1 isolated from a chicken in a broiler breeder flock in Queensland was used to experimentally infect 30 individually caged 22-week-old Cobb 500 broiler breeder hens. Another 10 birds were sham-inoculated with sterile broth. All birds failed to become colonized. At 29 weeks of age, all birds were transferred to a diet containing 50 parts/10 6 zinc bacitracin (ZnB) and were re-challenged with the same B. pilosicoli strain at 32 weeks of age, weekly for 5 weeks. The majority of the inoculated birds then became colonized, confirming previous findings that ZnB can increase susceptibility to colonization with B. pilosicoli. The control group remained uninfected. Infected groups tended to have an increased faecal water content and faecal staining of eggshells. Ten birds were then treated by crop tube with 25 mg/kg body weight tiamulin for 5 days, and 10 birds with 20 mg/kg body weight lincomycin for 5 days. Both treatments removed the infection, while untreated birds remained infected. The results support previous observations that ZnB at 50 parts/10 6 in the diet increases the susceptibility of birds to B. pilosicoli infection, and demonstrated the usefulness of both tiamulin and lincomycin for treatment of infection with B. pilosicoli in adult birds.     

Avian pathogenic Escherichia coli transmission from broiler breeders to their progeny in an integrated poultry production chain.

Early cases of colibacillosis with omphalitis, yolk sac infection and increased mortality were observed in five broiler chicken flocks (A1, A2, A3, A4 and B1) from two broiler breeder flocks A and B, respectively. Avian pathogenic Escherichia Coli (APEC) serotype O78, Fim/Tsh/Iuc pathotype, were isolated from flocks A, A1, A2, A3 and A4, and APEC serotype O139, pathotype Fim/Iuc, from flocks B and B1. APEC O78 strains isolated from broiler chicks A1, A2, A3 and A4, originating from breeder flock A, had the same antibiotic resistance pattern as APEC O139 strains isolated from broiler chicks B1 and breeder B.The random amplified polymorphic DNA technique performed on APEC strains revealed two distinct clusters of genetic similarity: cluster I consisted of some APEC O78 and cluster II of APEC O139. These results indicated that a transmission of APEC strains from adults A and B to their respective progeny could occur.     

Egg transmission of a respiratory isolate of Mycoplasma synoviae and infection of the chicken embryo

Egg transmission of Mycoplasma synoviae (Ms) was demonstrated in broiler breeder birds of over 40 weeks of age from a commercial flock that had been infected during rearing and in birds experimentally inoculated before onset of lay. Infection of growing birds with Ms did not prevent egg transmission in adult life. The embryonated eggs of experimentally infected hens were less susceptible to infection by inoculation than those of specific pathogen-free birds. Some chicks with maternal antibody were found to be infected at hatching and in these maternal antibody was not mycoplasmacidal. Indeed by protecting the embryo maternal antibody may have promoted hatching of infected chicks.     

Prevalence and disease association of intestinal spirochaetes in chickens in eastern Australia

Faecal samples (n = 1786) from chickens in broiler breeder (n = 28), layer (n = 22) or broiler (n = 19) flocks in the eastern states of Australia were cultured for intestinal spirochaetes. Overall, birds in 42.9% of broiler breeder and 68.2% of layer flocks were colonized with spirochaetes, but no birds in broiler flocks were infected. Colonization rates in infected flocks ranged from 10 to 100% of birds sampled. Faeces from colonized flocks were on average 14% wetter than those from non-colonized flocks. There was a highly significant association between colonization with spirochaetes and the occurrence of wet litter and/or reduced production. A subset of 57 spirochaete isolates from birds in 16 flocks were identified to the species level using a panel of polymerase chain reaction tests. Isolates from nine (56%) of these flocks were spirochaetes that are known to be pathogens of poultry: Serpulina pilosicoli was isolated from birds from five flocks, birds from two flocks were infected with Serpulina intermedia, and in two other flocks both species were identified. Isolates from the other seven flocks belonged to other Serpulina species, which are currently of unknown pathogenicity. This study indicates that infections with intestinal spirochaetes are a common but currently under-diagnosed cause of wet litter and/or reduced egg production in broiler breeder and layer flocks in Australia.     

Prevalence of antibodies to avian paramyxoviruses 1, 2 and 3 in wild and domestic birds in southern Spain

Haemagglutination inhibition tests were used to study the prevalence of antibodies to avian paramyxovirus (PMV) serotypes 1, 2 and 3 in wild and domestic birds in Andalusia (southern Spain). Tests were performed on 341 sera from layer hens drawn from 44 flocks, 198 from nine breeder hen flocks, 123 from five broiler chicken flocks, 329 from 47 turkey flocks, 51 from four pigeon flocks, 123 from five partridge flocks, 112 from five mixed domestic flocks and 361 sera from 24 wild species. The incidence PMV-1 antibodies was very low in wild species (1%), though higher in domestic birds (18%), due to the systematic vaccination of these species, particularly layer and breeder hens. The highest rate of infection for PMV-2 was found in domestic species (27%) and particularly in turkeys (47%). Among wild species, the house sparrow recorded the greatest prevalence of PMV-2 (69%). The PMV-3 infection rate was very low both in wild (6%) and domestic species; the figure of 15% recorded for the latter appears to be due to cross reacting PMV-1 vaccinal antibodies. The highest incidence in wild birds was recorded in the summer, whereas prevalence reached its maximum in domestic species in the winter.     

Molecular typing of avian pathogenic Escherichia coli colonies originating from outbreaks of E. coli peritonitis syndrome in chicken flocks

Escherichia coli colonies isolated from the bone marrow of fresh dead hens of laying flocks with the E. coli peritonitis syndrome (EPS) were genotyped using pulsed-field gel electrophoresis (PFGE). Typing is important from an epidemiological point of view and also if the use of autogenous (auto)vaccines is considered. Birds with EPS originated from one house of each of three layer farms and one broiler breeder farm. Farms were considered as separate epidemiological units. In total, six flocks were examined including two successive flocks of one layer farm and the broiler breeder farm. E. coli colonies (one per bird) from nine to 16 hens of each flock were genotyped. The clonality of E. coli within birds was studied using five colonies of each of nine to 14 birds per flock. E. coli genotypes, which totalled 15, differed between farms and flocks except for two successive layer flocks that shared three genotypes. One to five genotypes were found per flock with one or two genotypes dominating each outbreak. Within hens, E. coli bacteria were always clonal. Colonies of the same PFGE type always had the same multilocus sequence type. However, four PFGE types shared sequence type 95. Neither PFGE types nor multilocus sequence types were unambiguously related to avian pathogenic E. coli from EPS. In cases where persistence of E. coli strains associated with EPS is found to occur frequently, routine genotyping to select strains for autovaccines should be considered.     

Vertical transmission and clinical signs in broiler breeders and broilers experiencing adenoviral gizzard erosion

The present report documents an outbreak of adenoviral gizzard erosion in 22 broiler flocks in Germany. The clinical picture was characterized by uneven growth of affected broilers that resulted in considerably lower than average weight at slaughtering. Fowl adenovirus serotype 1 (FAdV-1) was isolated from gizzard lesions and histological examinations demonstrated FAdV-1-positive intranuclear inclusion bodies in gizzard epithelial cells of affected broilers by in-situ hybridization. Birds from all affected flocks originated from one broiler breeder farm. During production of affected birds, broiler breeders were between 27 and 32 weeks old. Enzyme-linked immunosorbent assay and specific virus neutralization assay of sera from parent birds demonstrated an acute FAdV-1 infection within the first 5 weeks of the production cycle. Clinically, broiler breeders exhibited a moderate fall in the hatchability of their chicks, while egg production remained normal. No further clinical signs could be observed. Genetically identical FAdV-1 strains were isolated from gizzards of embryos at the lowest point of hatchability and from affected broiler flocks raised on independent farms. For the first time, direct detection of viable FAdV-1 from gizzards of embryos and progenies of one FAdV-1-seropositive broiler breeder farm in the course of an outbreak of adenoviral gizzard erosion could be demonstrated, highlighting the importance of vertical transmission of this disease. Additionally, growth retardation and subsequent reduced average weight at the time of slaughter of broiler chickens underline the economic impact of adenoviral gizzard erosion for poultry production.     

Newcastle disease virus pathotypes

The clinical signs, mortality and postmortem findings following infection of six-week-old chicks with nine strains of Newcastle disease virus were studied. Although strains could be divided into four pathotypes the divisions were not clear-cut. The most prominent feature of disease following infection by two isolates from the post-1970 USA epidemic, a 1962 UK isolate and a 1972 UK isolate were haemorrhagic gut lesions. A virus isolate from the post-1970 UK epidemic, Lamb-Essex '70, rarely caused gut lesions in infected birds although this was the only strain to consistently induce oedema of the eye in infected birds.     

Experimental infection of broiler breeder hens with the intestinal spirochaete Brachyspira ( Serpulina ) pilosicoli causes reduced egg production

The pathogenic potential of the anaerobic intestinal spirochaetes Brachyspira ( Serpulina ) pilosicoli and Brachyspira innocens was evaluated in adult chickens. Thirty 17-week-old Cobb broiler breeder hens were individually caged in three groups of 10 birds. Control birds (group A) were sham inoculated with sterile broth medium. Birds in the other two groups (groups B and C) were inoculated, respectively, with an isolate of B. innocens or of B. pilosicoli . Birds were monitored daily, and killed at 41 weeks of age. Infection had no consistent effect on body weight gain, but inoculation with B. pilosicoli resulted in a transient increase in faecal water content. B. innocens infection had no effect on egg production, but B. pilosicoli infection caused a delayed onset of laying, and a highly significant reduction in egg production over the first 11 weeks of lay. This study confirms that B. pilosicoli can cause serious egg production losses in adult chickens, while B. innocens is not obviously pathogenic.     

Mycoplasma iowae infection in young Turkeys

A comparison was made of the effect of five different strains of M. iowae after inoculation of one-day-old poults via the thoracic air sac and foot pad. Three strains appeared to be more virulent and more invasive than the other two, causing stunting, poor feathering and leg abnormalities including chondrodystrophy. One of these three strains was used in a second experiment in which three routes of infection were compared. Infection in ovo caused a severe generalised disease in hatched poults with high mortality. The only two birds which survived into the third week developed chondrodystrophy. One group was infected orally at one day of age and some birds developed bone and joint abnormalities but another group, infected via both the thoracic air sac and the foot pad, also at one day of age, developed a higher incidence of these, abnormalities, which included chondrodystrophy, rotated tibia, deviated toes and, in a few cases, erosion of the articular cartilage of the hock joint. Some of the control uninfected birds developed leg abnormalities but never chondrodystrophy, rotated tibia or cartilage erosion. M. iowae was most widely disseminated in tissues following in ovo infection and least after oral infection. Isolations became less frequent with age and no organisms were recovered in birds sampled at 12 weeks. In neither experiment could antibodies to M. iowae be detected by rapid agglutination.     

Effects of experimental immunosuppression on reovirus-induced tenosynovitis in light-hybrid chickens

Groups of specific pathogen-free light-hybrid chickens which had been immunosuppressed either by surgical thymectomy (Tx) or surgical bursectomy (Bx) or cyclophosphamide (Cy) treatment or Tx plus Cy treatment (Tx + Cy), as well as intact (untreated) birds, were inoculated with graded doses of an arthrotropic avian reovirus at 1 day of age and observed up to 5 weeks post-inoculation (p.i.). Cy-treatment with or without Tx considerably increased the mortality, incidence of gross leg lesions and severity of microscopic lesions due to reovirus infection. The Bx group showed only a significant increase in mortality, and the Tx group response was generally similar to the untreated group. Dead birds showed hepatic necrosis, which in Cy-treated groups (Cy, Tx + Cy) was associated with calcification. Surviving Cy-treated birds had acute tenosynovitis characterised grossly by large amounts of serous exudate in leg tendon sheaths, and microscopically by a massive heterophilic but only mild lymphocytic infiltration of tendon sheaths. Tenosynovitis lesions in Bx birds were generally similar to those of the untreated chickens, i.e. grossly small amounts of yellowish brown gelatinous exudate and microscopically moderate chronic inflammatory changes in leg tendon sheaths. In Tx birds gross lesions were rarely seen and the microscopic lesions were often very mild. Reovirus could be recovered from cloacal swabs from untreated and Tx birds for 2 weeks, Bx birds for 3 to 4 weeks, and Cy and Tx + Cy chickens continuously throughout. Reovirus was isolated from tendon tissue of all Cy and Tx + Cy infected birds examined at 5 weeks p.i. and gross tenosynovitis lesions were seen in all birds. The virus was recovered from the tendons of only a proportion of the infected untreated, Tx and Bx groups, and overall more frequently from apparently normal birds. This was especially marked in the infected Tx group. Antibody responses as shown by gel precipitation and virus neutralisation were positive and similar in untreated and Tx birds, were delayed in the Bx group with the precipitation test only, and absent from most of the Cy and Tx + Cy birds. The results of these experiments indicate that recovery from reovirus infection probably involves both B- and T-cell systems but that the B-cell system is predominantly protective.     

Genetic diversity and phylogenetic analysis of glycoprotein gp85 of avian leukosis virus subgroup J wild-bird isolates from Northeast China

Avian leukosis virus subgroup J (ALV-J), first isolated in 1989, preferentially infects meat-type birds. Chinese layer flocks have experienced outbreaks of this virus since 2008. To analyze the status of ALV-J infection in wild birds in China, 585 wild birds collected from three provinces of Northeast China from 2010 to 2012 were tested, and six ALV-J strains were isolated for the first time. Furthermore, the gp85 genes of the six strains were amplified, cloned, and sequenced. The results indicated that two different ALV-J strains coexisted in Chinese wild birds from 2010 to 2012. These results not only expand the epidemiological data available for ALV-J and provide necessary information for the further understanding of the evolution of ALV-J, but they also highlight the potential role of wild-bird migration in the spread of ALV-J.     

Pathogenicity of four strains of staphylococci isolated from chickens with clinical tenosynovitis

Four strains of staphylococci of different phage patterns (two principal and two minor phage types) associated with naturally occurring tenosynovitis were studied for their virulence for chickens and tropism for tendon tissue. There were significant differences in virulence between the principal and minor phage types of Staphylococcus aureus. Infection with the principal phage types produced a generalised septicaemic disease and no specific tropism of the bacteria for tendon tissue although there were gross and histopathological changes in tendons and tendon sheaths indistinguishable from those in naturally occurring tenosynovitis. However, the patho-genesis of the disease observed in the experimental infections was different to that considered to occur in natural cases of tenosynovitis and provided further indirect evidence for the secondary nature of the staphylococcal infection in tenosynovitis.     

Organ culture studies on adenoviruses isolated from tenosynovitis in chickens

Five strains of adenovirus isolated from tenosynovitis in chickens were examined for their ability to grow in vitro in pieces of explanted tendon from 20-day-old chicken embryos from light or heavy breeds. Replicate tendon organ cultures (TOCs) were infected with three concentrations of each strain of virus and supernatant fluids were assayed for virus at intervals after infection up to 11 days. Two strains grew well in TOC, one moderately well, and the other two failed to grow. A strain of Newcastle disease virus (NDV), an agent not associated with tenosynovitis, also failed to grow. TOC from broiler embryos seemed slightly more susceptible to the two actively growing viruses than explants from a light breed. Growth of the adenovirus was demonstrated by immuno-fluorescence staining of sections of TOC but the appearance of stained antigen was dependent on virus titre. One of the strains which showed no activity in TOC grew in tracheal organ cultures (TrOC), as did one which multiplied in tendon, and NDV. In a more prolonged experiment, one of the viruses was found to persist in TOC at high titre for at least 37 days. The likelihood of the in vitro behaviour of these adenoviruses reflecting that occurring in vivo in tendon tissue of infected chickens is discussed.     

Viral arthritis/tenosynovitis: a review

A review if presented of viral arthritis/tenosynovitis, one of the clinical manifestations of avian reovirus infection in chickens. Since the detection of a viral etiology of arthritis/tenosynovitis in 1957, the disease has been reported in various parts of the world. Pathological features of the disease include inflammatory lesions in the extensor and flexor tendons and tendon sheaths of the posterior limbs and in the tibiotarsal joints. Hepatitis, myocarditis, hydropericardium, as well as intestinal and respiratory tract involvement are described in association with avian reovirus infection. The epizootiology, horizontal and vertical transmission of reovirus, as well as the role of maternal immunity and its application in breeder vaccination for the control of viral arthritis/tenosynovitis are described.     

Epidemiological and pathological studies of subgroup J avian leukosis virus infections in Chinese local "yellow" chickens.

Epidemiological, pathological and molecular studies indicate that subgroup J avian leukosis virus (ALV-J) infections are widely spread in "yellow chickens" of local breeds in China. ALV-J induced tumour mortality and the serological conversion rates to ALV-J were very high in some breeder flocks. Typical myelocytomatosis was demonstrated not only in livers, spleens, kidneys, and sternums, as in white meat-type chickens, but also in thymuses and the bursa of Fabricius. Especially, severe myeloid cell infiltration was found throughout the whole enlarged thymuses of some birds. ALV-J was isolated at high positive rates from both liver tumour samples and embryos collected from breeder flocks with tumours. At the same time, reticuloendotheliosis virus was also co-isolated with ALV-J in some tumour samples and embryos. Sequence analysis of env genes demonstrated that the gp85 and gp37 among six ALV-J isolates from "yellow chickens" of Chinese local breeds varied as highly as among ALV-J strains isolated from white meat-type chickens worldwide. But strain GD0512 isolated in 2005 from a "yellow chicken" farm in southern China had high identity of 95.1% for gp85 or 99.5% for gp37 to strain HN0001 isolated in 2000 from a white meat-type breeder farm in northern China, a much higher identity than to other yellow chicken and white chicken strains. This is the first report of the isolation and identification of ALV-J from yellow chickens of Chinese local breeds and also the first report of vertical co-infection of ALV-J and reticuloendotheliosis virus. The significance of co-infection of ALV-J and reticuloendotheliosis virus in pathogenesis is discussed.     

Swollen head syndrome in Taiwan-isolation of an avian pneumovirus and serological survey

Outbreaks of swollen head syndrome (SHS) were observed in two broiler and two broiler-breeder farms in Taiwan. The disease was characterized by oedematous swelling of the head, especially surrounding the eyelids, the neck and wattles. Avian pneumovirus and Escherichia coli were isolated from birds in all four farms. In addition, Staphylococcus aureus and infectious bursal disease virus were each isolated from one farm. A serological survey of 398 birds from 11 broiler breeder farms showed 86.4% (344) of them had ELISA antibodies against turkey rhinotracheitis virus.     

Association of the chicken MHC B haplotypes with resistance to avian coronavirus

Clinical respiratory illness was compared in five homozygous chicken lines, originating from homozygous B2, B8, B12 and B19, and heterozygous B2/B12 birds after infection with either of two strains of the infectious bronchitis virus (IBV). All chickens used in these studies originated from White Leghorn and Ancona linages. IBV Gray strain infection of MHC homozygous B12 and B19 haplotype chicks resulted in severe respiratory disease compared to chicks with B2/B2 and B5/B5 haplotypes. Demonstrating a dominant B2 phenotype, B2/B12 birds were also more resistant to IBV. Respiratory clinical illness in B8/B8 chicks was severe early after infection, while illness resolved similar to the B5 and B2 homozygous birds. Following M41 strain infection, birds with B2/B2 and B8/B8 haplotypes were again more resistant to clinical illness than B19/B19 birds. Real time RT-PCR indicated that infection was cleared more efficiently in trachea, lungs and kidneys of B2/B2 and B8/B8 birds compared with B19/B19 birds. Furthermore, M41 infected B2/B2 and B8/B8 chicks performed better in terms of body weight gain than B19/B19 chicks. These studies suggest that genetics of B defined haplotypes might be exploited to produce chicks resistant to respiratory pathogens or with more effective immune responses.     

Field studies on the association between amyloid arthropathy and Mycoplasma synoviae infection,and experimental reproduction of the condition in brown layers

Approximately 20% of outbreaks of amyloid arthropathy in The Netherlands were earlier attributed to Enterococcus faecalis but there was no explanation for the remaining cases. In a further study, material from the joints of 10 birds from each of 10 affected brown layer flocks and two broiler flocks was cultured in several bacteriological media. E. faecalis was isolated from one layer flock but Mycoplasma synoviae was recovered from purulent joints in six of the layer flocks and both broiler flocks. Brown layers were then infected experimentally with one of the M. synoviae isolates to assess its arthropathic and amyloidogenic potential. Using Congo red staining, articular amyloid deposits were seen 12 weeks after intra-articular or intravenous inoculation in almost all birds. After intra-articular inoculation of the left knee joint the contralateral hock tendon sheath tended to be affected, while hock and foot joints were mainly affected after intravenous inoculation. Amyloid deposits were seen in livers and spleens of many birds. This is the first demonstration of arthropathic strains of M. synoviae with an association with amyloid arthropathy, while for one isolate its amyloidogenic potential was shown experimentally.