Occurrence of Mycoplasma gallisepticum and avian metapneumovirus in commercial broiler flocks from the Southeast and Midwest regions of Brazil

From 50 commercial broiler flocks included in a study concerning respiratory disease, signs of swollen head syndrome (SHS) were shown in eight. Postmortem examination was performed in eight birds showing signs of SHS from each flock. The trachea and head from each bird were collected for laboratory investigation. An enzyme-linked immunosorbent assay (ELISA) was used for the detection of viral and avian mycoplasma antigens in the trachea, and bacteriologic examinations were performed from the infraorbital sinuses of the infected birds. According to the ELISA results, the most frequently detected antigen in the trachea was Mycoplasma synoviae (six flocks, 75%), followed by infectious bronchitis virus (IBV) (five flocks, 62.5%), avian adenovirus (four flocks, 50%), avian reovirus (three flocks, 37.5%), Mycoplasma gallisepticum (one flock, 12.5%), and Newcastle disease virus (NDV) (one flock, 12.5%). Turkey rhinotracheitis (TRT), infectious laryngotracheitis, and avian influenza viral antigens were not detected. Experimental assays for characterization of NDV and IBV isolates showed that they were strains of low virulence (evidently vaccine strains). Bacteriologic examinations from the infraorbital sinuses of the affected birds resulted in the isolation of Escherichia coli (seven cases, 87.5%) and Staphylococcus spp. (one case, 12.5%). It is evident that TRT virus did not play a causal role in SHS in commercial broiler flocks in Greece, but in this condition, other viruses (IBV, NDV), mycoplasmas, or bacteria may be involved, and environmental conditions seem to be essential to the occurrence and severity of the disease.

Aim:The present study was designed for the detection of the most prevalent respiratory infections in chicken flocks and clarifying their interaction and impact on flock health.Materials and Methods:A total of 359 serum samples were collected from 55 backyard chickens and tested using commercial enzyme-linked immunosorbent assay kits to determine the seroprevalence of Newcastle disease virus (NDV), infectious bronchitis virus (IBV), influenza type A, Mycoplasma gallisepticum (MG), and Mycoplasma synoviae (MS). Molecular prevalence of NDV, IBV, low pathogenic avian influenza virus (LPAIV) H9N2, MG, and MS was carried out on swab, and tissue samples collected from 55 backyard flocks and 11 commercial broiler flocks suffered from respiratory infections using polymerase chain reaction (PCR) and reverse transcription-PCR.Results:Seroprevalence of NDV, IBV, Influenza type A virus, MG, and MS in chicken backyard flocks was 56.4%, 50.9%, 12.7%, 14.5%, and 3.6%, respectively. Specific antibodies against one or more respiratory viruses and mycoplasma were detected in 36.4% of backyard flocks, indicating concurrent viral infections. The molecular survey showed that 90.9% of chicken backyard flocks were infected with common respiratory viruses (NDV, IBV, and LPAIV H9N2) while 81.8% of commercial broiler flocks were infected. The molecular prevalence rate of NDV, IBV, and LPAIV H9N2 was 46.97%, 56.1%, and 19.7% in backyard flocks, respectively. Combined viral and bacterial infection represented 40% and 63.6% of the respiratory infections, resulting in enhanced pathogenicity and increased mortalities of up to 87.5% and 27.8% in backyard and commercial flocks, respectively. Mixed infection of IBV, LPAIV H9N2, and/or Escherichia coli is the most prevalent mixed infection in broiler flocks, inducing severe clinical outcomes. Avian pathogenic E. coli was, respectively, isolated from 40% of backyard flocks and 81.82% of broiler flocks. Staphylococcus aureus was isolated from three backyard chicken flocks mixed with other respiratory pathogens with elevated mortality. Mixed infection of E. coli and MG reported in 9.1% of broiler flock. MG was detected in 14.5% of backyard flocks and 9.1% of broiler flocks while MS was detected only in 3.6% of backyard chickens mixed with E. coli, and other viruses.Conclusion:Our results confirm that mixed infections are more commonly prevalent and associated with dramatic exacerbation in clinical outcomes than a single infection. Bidirectional synergistic interaction between these concurrently interacted respiratory pathogens explains the severe clinical impact and high mortality rate. The high prevalence of IBV (either as a single or combined infection) with LPAIV H9N2 and/or E. coli, in spite of intensive use of commercial vaccines, increases the need for revising vaccination programs and the application of standard biosecurity measures. Backyard chickens impose a great risk and threaten commercial flocks due to the high prevalence of viral respiratory pathogens.

In recent years, the impact of respiratory disease resulting from Avian Metapneumovirus (aMPV) infection has been generally rising in the broiler industry in Europe. In this context, in order to investigate aMPV contribution to the clinical picture and the potential benefits of diversified vaccination strategies compared to nonvaccination policies, a longitudinal monitoring was performed, also evaluating Infectious Bronchitis Virus (IBV) presence. Broiler flocks located in Western France, where aMPV has already proven to be a health and productivity issue, were screened by RT-PCR on rhino-pharyngeal swabs, and the viruses were genetically characterized by sequence analysis. For a more comprehensive picture of aMPV molecular epidemiology and evolution in France, aMPV subtype B strains detected from 1985 to 1998 were sequenced and included in the analysis. The survey confirmed the detection of aMPV subtype B in commercial broiler flocks in France, together with a certain heterogeneity demonstrated by the circulation of more recent and historical French field strains. No IBV field strains were detected. The implementation and evaluation of different management choices and vaccine strategies suggests once again that immunization does not prevent infection but contributes greatly to the containment of the clinical manifestations.

Avian metapneumovirus causes an acute highly contagious upper respiratory tract infection primarily of turkeys and chickens. The disease can cause significant economic losses in turkey and chicken flocks, particularly when exacerbated by secondary pathogens. The purpose of this study was to determine the prevalence of avian metapneumovirus antibodies in broiler and broiler breeder flocks in Kermanshah province, west of Iran. All the flocks had not been vaccinated against avian metapneumovirus. The province were divided into four geographic areas; southwest, southeast, northwest, and northeast. Flocks in each area, and 14–15 birds in each flock, were randomly sampled. The blood samples were taken regardless of the presence of any signs of respiratory or any other clinical disease in the flocks. A total of 435 blood samples were collected from 30 commercial chicken flocks (24 broiler flocks, aged between six and eight weeks, and six broiler breeder flocks, aged between 56 and 72 weeks). The presence of antibodies against avian metapneumovirus in each serum sample was tested twice by enzyme-linked immunosorbent assay using a commercial kit which was able to determine antibodies against A, B and C subtypes of avian metapneumovirus. Out of 347 serum samples obtained from broiler chickens, 167 (48.1%) were positive to avian metapneumovirus antibodies, which represented 20 (83.3%) of 24 examined broiler flocks. Out of 88 samples obtained from broiler breeder chickens, 82 (93.2%) were positive to avian metapneumovirus antibodies, which belonged to six (100%) of examined broiler breeder flocks. Detection of anti-avian metapneumovirus antibodies among broiler breeder (100%) was higher than broiler (83.3%) flocks. A higher rate of seropositivity (83.3% of samples and 100% of broiler flocks) was observed in northwest. The results of this study may indicate the possible involvement of avian metapneumovirus in the respiratory disease we are seeing in chickens in Iran. Its prevalence has to be investigated in other parts of Iran. Future work may and should include the use of molecular methods and isolation of the virus. Isolation of avian metapneumovirus will allow the possibility of making autogenous vaccines.

Molecular Survey of Avian Respiratory Pathogens in Commercial Broiler Chicken Flocks with Respiratory Diseases in Jordan

Lameness assessment with automatic monitoring of activity in commercial broiler flocks

Body weight, feed-particle size, and ascites incidence revisited

A total of 4,111 chicken sera collected from 1985 to 1995 at 137 farms in 36 prefectures were subjected to an enzyme-linked immunosorbent assay (ELISA) to test for antibodies against turkey rhinotracheitis (TRT) virus. The antibodies to TRT virus were detected in the chicken sera collected from 1988 to 1995, but the antibodies to TRT virus were undetectable in the sera of chickens collected before 1987. The antibody positive rates each year ranged from 2.2% (1988) to 54.2% (1995). Recently, the TRT virus has spread over the flocks of commercial broiler and layer chicken throughout the country. The serological findings indicated that the TRT virus had invaded the chickens before 1988, and has been widespread thereafter in Japan.

Infection of poultry with Eimeria spp., the causative agent of coccidiosis, can predispose birds to necrotic enteritis (NE) caused by netB gene-positive strains of Clostridium perfringens. The detection of Eimeria spp., C. perfringens, and netB were examined in settled dust from broiler flocks under experimental and field conditions. Dust samples were collected from settle plates twice weekly from two experimental flocks inoculated with three species of pathogenic Eimeria in 9-day-old chicks, followed by netB gene-positive C. perfringens 5 days later to produce subclinical and clinical NE. A noninoculated flock was sampled weekly from day 0 and served as a control flock. An additional 227 dust samples from commercial broiler flocks were collected at the end-of-batch (6-7 wk of age; one scraped dust sample per flock). In the NE-subclinical and NE-clinical flocks, high levels of Eimeria spp. and C. perfringens were detected after inoculation followed by a gradual decline over time. In the control flock, C. perfringens and netB were detected at low levels. No significant effect of sampling location was evident on Eimeria spp., C. perfringens, and netB load within poultry houses. These results provide evidence that Eimeria spp., C. perfringens, and netB gene copies can be readily measured in poultry dust samples collected in settle plates and may provide an alternative sampling method for monitoring flock coccidiosis and NE status. Further studies are required to assess the utility for such a test in commercial flocks.

Recent studies have indicated that crop contamination increases during preslaughter feed withdrawal and that contaminated crop contents may serve as an important source of Salmonella entry into poultry processing plants. During the present study, we evaluated the effect of preslaughter feed withdrawal on crop pH and Salmonella crop contamination in broilers from three commercial broiler flocks. The effect of experimental feed withdrawal on crop pH, lactic acid concentration, and Salmonella crop contamination was also evaluated in market-age broilers challenged experimentally with Salmonella typhimurium. Crop pH increased significantly (P < 0.05) from 3.64 +/- 0.25 before feed removal to 5.14 +/- 0.72 after 8 hr of feed withdrawal in broilers from commercial flocks. The incidence of Salmonella crop contamination in the commercial broilers increased (P < 0.05) from 3.3% before feed removal to 12.6% after 8 hr of feed withdrawal. Similarly, crop pH increased (P < 0.05) by a magnitude of approximately 1 unit in broilers after 8 hr of experimental feed withdrawal. The population of S. typhimurium in the crops of the experimentally challenged broilers increased (P < 0.05) by approximately 1 log unit during the 8-hr experimental feed withdrawal. The concentration of lactic acid in the crop of the broilers during experimental feed withdrawal decreased (P < 0.01) from a range of 119-135 mumol/ml before feed removal to a range of 22-32 mumol/ml after 8 hr of feed withdrawal. The results indicated that feed withdrawal resulted in a decrease in lactic acid in the crop, accompanied by an increase in crop pH, and an increase in Salmonella crop contamination.

Genotype dynamics of Campylobacter jejuni in a broiler flock

Identifying Eimeria spp. circulating in a poultry flock assists in designing vaccine preventive programs, as different species do not cross-protect. Because species differ in anticoccidial drug susceptibility, species identification can also be used to optimize anticoccidial medication. In the present study, we designed pan-Eimeria-specific primers for the 18S rDNA and the cytochrome oxidase I (COI) genes, and tested whether next-generation sequencing of their amplicons allowed reliable identification of Eimeria spp. in samples of isolated oocysts. For each gene, two sets of primers to be used in a nested PCR (nPCR) system were designed. In silico evaluation of the primers using published sequences showed that nucleotide sequence identities of the nested amplicons were less than 97% between most species, while only identities of 18S rDNA genes of Eimeria necatrix and Eimeria tenella and between the COI genes of Eimeria mitis and Eimeria mivati were higher than 97%. Three vaccines and five Eimeria samples from chickens in backyard flocks were investigated by nPCRs and by direct PCRs (dPCR) using the nested (inner) primers with genomic DNA as the template. Seventeen further Eimeria samples from chickens in backyard flocks and three Eimeria samples from commercial broiler flocks were investigated only by nPCR. Sequencing nPCR products tended to detect more species than sequencing dPCR products and sequencing 18S rDNA products tended to detect more species than sequencing COI products. Regarding the detected species, there was a clear difference between the commercial broiler flocks and the backyard flocks. Eimeria acervulina, Eimeria maxima, and E. tenella/E. necatrix were the only species detected in broiler flocks, while the population in the backyard flocks was more varied, with Eimeria brunetti and E. mitis/E. mivati and the previously described operational taxonomic unit Y being more prevalent. Several sequences having less than 97% identity with one of the sequences used for clustering were detected in samples from backyard flocks. In conclusion, next-generation amplicon sequencing can be a useful tool to determine which Eimeria spp. are circulating in chicken flocks.

The present study was undertaken to investigate the incidences of nephropathy encountered in young chicks of certain commercial broiler flocks in the states of Telangana, Andhra Pradesh, Tamil Nadu and Maharashtra. The mortality and growth retardation associated with the condition was a major concern, necessitating a thorough study to establish the etiology. Detailed necropsy of the dead chicks revealed marked inflammation of the kidneys along with gout. Mortality percentage recorded was up to 20%. Samples were collected from the affected flocks for isolation and identification of the causative agent. The presence of ANV RNA was detected using reverse transcriptase-polymerase chain reaction and 42 kidney samples (43.75%) were found to be ANV positive while all were IBV negative. The positive samples were successfully propagated in Specific Pathogen Free chicks and embryonated eggs as was evident from the changes observed and further confirmation using RT PCR. The nucleotide sequence of the representative ANV isolate was determined and showed high identity with ANV sequences in the NCBI database. In the constructed phlyogenetic tree, the ANV isolates were clustered together distinctly separate from the related Chicken Astro virus. This investigation confirms the involvement of ANV in cases of gout, nephritis and related mortality in commercial broiler flocks.

This study reports the co-existence of two S1 mutants of GI-13 (4/91-like) Infectious Bronchitis Virus (IBV) and Mycoplasma gallisepticum (MG) in a backyard poultry flock that had non-vaccinated 30 broiler chickens and four turkey pullets. Serum samples and tracheal swabs were taken from the chickens and turkey pullets showing respiratory signs. Serum antibody levels were measured using commercial ELISA kits against IBV, Avian Influenza Virus (AIV), Newcastle Disease Virus (NDV), Avian Metapneumovirus (AMPV), MG, Mycoplasma synoviae (MS), and Ornithobacterium rhinotracheale (ORT). Additionally, tracheal swabs were tested for AIV serotypes H5, H7, and H9, NDV, IBV, AMPV, MG, MS, Pasteurella multocida, Avibacterium paragallinarum, and Bordetella avium by circular amplification technology (CAT). Anti-MS, -IBV, -MG, -NDV, -AMPV, and -ORT IgG antibodies were detected in some chicken sera, while anti-NDV, -MG, -MS, and -ORT IgG antibodies were detected in turkey sera. All avian tracheal swabs were positive for MG. However, IBV was only detected in chicken tracheal samples tested by CAT. The IBV strains were genotyped by sequencing a part of the S1 glycoprotein gene. The alignment analyses of two isolates showed 99.35% and 98.69% nucleotide similarities and 99.02% amino acid similarities with the 4/91 IBV vaccine and field strains. Two mutants showed 99.35% nucleotide and 100% amino acid sequence identity to each other. The turkeys and chickens in the flock had MG and MG/IBV co-infections, respectively. Consequently, the presence of mutants of 4/91 (GI-13) IBV genotypes and MG found in backyard poultry could be a potential epidemiological source for commercial flocks in poultry integrations.

A U.S. isolate of avian pneumovirus (APV), APV/MN/turkey/1-a/97, was attenuated by serial cell culture passages in chicken embryo fibroblasts (seven passages) and Vero cells (34 passages). This virus was designated as APV passage 41 (P41) and was evaluated for use as a live vaccine in commercial turkey flocks. The vaccine was inoculated by nasal and ocular routes in 2-to-4-wk-old turkeys in 10 turkey flocks, each with 20,000-50,000 birds. Only 2 birds per 1000 birds were inoculated in each flock with the expectation that bird-to-bird passage would help spread the infection from P41-exposed birds to their respective flock mates. The virus did spread from vaccinated birds to the entire flock within 10 days as detected by reverse transcription-polymerase chain reaction. Mild respiratory illness was observed in a few birds 12 days postvaccination in 2 of 10 flocks. Within 3 wk postvaccination, all flocks became seropositive for APV antibodies as measured by enzyme-linked immunosorbent assay. In an additional flock, the virus was administered to all turkeys simultaneously in drinking water and seroconversion occurred within 2 wk. All 11 flocks remained seropositive until 10 wk postvaccination. When compared with unvaccinated flocks on the same farm from the previous year, the medication cost, total condemnation, and mortality rates attributed to APV were lower in P41-vaccinated flocks. When birds from vaccinated flocks were challenged with virulent APV under experimental conditions, no clinical signs were observed at 2, 6, and 10 wk postvaccination, whereas in the control unvaccinated birds, respiratory illness and virus shedding occurred after challenge. These results indicate that P41 administered by the nasal and ocular routes, and by drinking water, causes seroconversion and induces protection from virulent APV challenge for at least 10 wk.