Except for the important role coccidia have as predisposing factors of necrotic enteritis, the role parasites play in the dynamics of a healthy microbiota of chickens is not well explored. This review describes the interactions of relevant intestinal parasites of chickens with bacteria.

Infection with Eimeria spp. favor the growth of Clostridium perfringens and suppress the growth of many other bacteria by increasing viscosity and passage time of the ingesta, and by causing lesions to the intestinal mucosa that improve the availability of nutrients for C. perfringens. Conversely, there are indications that bacteria influence the course of disease after infections with Eimeria spp. Not much is known about intestinal cryptosporidiosis in chickens, but results in mice show that the intestinal microbiota induces some resistance against infection with Cryptosporidium parvum and that the innate immune response triggered by infections with cryptosporidia might have an effect on other intestinal microbes. Histomonas meleagridis depend on bacteria in vitro, and in vivo it will cause lesions in chickens only in the presence of bacteria. Blastocystis spp. are very common in chickens, but there is no information about interactions with bacteria. In humans, there is evidence of the correlation of the detection of Blastocystis and changes in the intestinal microbiota. There are indications of interactions between Ascaridia galli and various bacteria in chickens and Ascaridia spp. of mammals are known to produce various types of antimicrobial molecules. However, often the underlying mechanisms of these interactions between parasites and bacteria remain unknown and only correlations but not causation can be established.

After recovery, house finches (Haemorhous mexicanus) reinfected with the same Mycoplasma gallisepticum strain remain partially resistant to reinfection for at least 14 mo in that they recover from reinfection much more rapidly than do Mycoplasma gallisepticum-naïve birds. To test the response of birds to reinfection with a heterologous strain we performed two experiments. In a first experiment we exposed birds to one of three strains that differed in virulence. After they had recovered all were reinfected with the most virulent-strain available at the time of the experiment. In a second experiment we infected and later reinfected house finches with one of two Mycoplasma gallisepticum strains whereby we switched the order of the strain used. In both experiments, disease in birds reinfected with a more-virulent strain caused more-severe disease. Our data suggest that the observed increase in Mycoplasma gallisepticum virulence, once the disease has become endemic in free-ranging house finches is—in part—driven by increased resistance of recovered birds to strains of equal or lower virulence.

Infectious bronchitis virus (IBV) is one of the major poultry pathogens of global importance. However, the prevalence of IBV strains in Malaysia is poorly characterized. The partial genomic sequences (6.8 kb) comprising the S-3a/3b-E-M-intergenic region-5a/5b-N gene order of 11 Malaysian IBVs isolated in 2014 and 2015 were sequenced using next-generation sequencing technology. Phylogenetic and pairwise sequence comparison analysis showed that the isolated IBVs are divided into two groups. Group 1 (IBS124/2015, IBS125/2015, IBS126/2015, IBS130/2015, IBS131/2015, IBS138/2015, and IBS142/2015) shared 90%–95% nucleotide and deduced amino acid similarities to the QX-like strain. Among these isolates, IBS142/2015 is the first IBV detected in Sarawak state located in East Malaysia (Borneo Island). Meanwhile, IBV isolates in Group 2 (IBS037A/2015, IBS037B/2015, IBS051/2015, and IBS180/2015) were 91.62% and 89.09% identical to Malaysian variant strain MH5365/95 (EU086600) at nucleotide and amino acid levels, respectively. In addition, all studied IBVs were distinctly separate from Massachusetts (70%–72% amino acid similarity) and European strains including 793/B, Italy-02, and D274 (68%–73% amino acid similarity). Viruses in Group 1 have the insertion of three amino acids at positions 23, 121, and 122 of the S1 protein and recombinant events detected at nucleotide position 4354–5864, with major parental sequence derived from QX-like (CK-CH-IBYZ-2011) and a minor parental sequence derived from Massachusetts vaccine strain (H120). This study demonstrated coexistence of the IBV Malaysian variant strain along with the QX-like strain in Malaysia.

Three studies were conducted using Clostridium perfringens as an intestinal challenge to produce necrotic enteritis (NE). The studies consisted of two battery screening studies and one production study in floor pens. The purpose of the trials was to determine if reducing the level of microorganisms in feed consumed by broilers reduced the impact of a nonfeed-based Clostridial challenge. In all studies, C. perfringens challenged broilers consuming feed containing lower levels of microorganisms compared to control feed exhibited significantly (P < 0.05) better feed conversion (feed conversion was improved by 14% in battery trials and by 4.2% in the pen trial) than did C. perfringens-challenged broilers consuming control feed. In battery trials, body weight gain and NE-associated mortality were also significantly improved in C. perfringens-challenged broilers consuming feed containing lower levels of microorganisms (16.5% improvement in body weight gain and 72.5% reduction in NE-associated mortality). In the pen trial, body weight gain and NE-associated mortality appeared unaffected by feed microbial quality. No effect was observed on lesion scores. The present data indicate that reducing the level of microorganisms in feed can ameliorate some of the performance losses associated with a Clostridia challenge.

Fowl cholera (FC) is a highly contagious and economically important disease of poultry worldwide. This study was performed on 218 Pasteurella multocida isolates collected from separated breeding farms or backyards with acute and chronic FC cases in multiple localities across Hungary during the period 2005–2010. All isolates were characterized by a broad range of biochemical, serological, and molecular methods, as well as their antibiotic susceptibility to aminoglycosides (A), macrolides (M), penicillins (P), quinolones (Q), cephalosporins, sulphonamides (S), and tetracyclines (T) was determined. Fifty-two percent of all isolates belonged to a well-defined type that was highly virulent, caused acute FC, and had the same character: fermented L-arabinose, possessed capsule type A, identified as Heddleston serotype 1, and possessed allele type A of the ptfA fimbrial gene. This type was widely distributed among poultry in Hungary, especially in waterfowl flocks.

Isolates collected from the chronic FC cases were more diverse: none of them fermented L-arabinose; they possessed capsule type A (76%), F (9%), or was non-typeable (15%) with different Heddleston serotypes, mainly 1, 3, 4, and 5, or 7 and 16; in addition, possessed allele type B of ptfA fimbrial gene. Only 26 isolates presented characters similar to any of the chronic FC cases but caused severe disease.

The antibiotic susceptibility assay presented that 80% of all isolates were resistant to 1–5 of the studied antimicrobial agents. During the survey, after two years, there was a dramatic decline both in the number of the multi-drug resistance phenotypes and the prevalence of the highly virulent type of the isolates. In the next four years, multiresistant isolates were almost completely removed, whereas the number of isolates resistant to 1 or 2 drugs was constant.

Reduced frequency of antibiotic multiresistant, mostly L-arabinose-fermenting isolates, has been observed since 2007. This reduction may be a consequence of the elimination of multiple waterfowl flocks in Hungary during avian influenza outbreaks, which possibly created a break in the “transmission chain” of pathogenic P. multocida isolates.

The ability of commercial vaccines H120 and 4/91 to protect against Moroccan-Italy 02 infectious bronchitis virus (Mor-It02) was investigated in specific-pathogen-free (SPF) chickens and commercial broiler chickens. Commercial broiler chicks (Experiment 1) were vaccinated at the hatchery with H120 vaccine at Day 1, and challenged at Day 21 with 10⁴ 50% egg-infective dose (EID₅₀) of Mor-It02. All chicks were observed daily for clinical signs attributable to Mor-It02 infection during the 10 days postchallenge (pc). At 5 and 10 days pc, chicks were humanely sacrificed for necropsy examination, and tissues were collected for histopathology evaluation. To better understand the findings on commercial broilers, day-old SPF chicks were divided into five groups in a second experiment: Group Mass/4-91, vaccinated with H120 and 4/91 respectively at Days 1 and 15 of age; Group Mass/Mass, vaccinated by H120 at Days 1 and 15; Group Mass, vaccinated with H120 at Day 1; Group NV, kept unvaccinated; and Group NC, kept as a negative control (unchallenged). At Day 24 of age, Groups Mass/4-91, Mass/Mass, Mass, and NV were challenged with 10⁴ EID₅₀ of Mor-It02. In both experiments, blood samples were collected at different periods for serologic analyses. Oropharyngeal swabs were collected for virus detection by reverse-transcription PCR. In Experiments 1 and 2, respiratory signs started as early as 24 hr pc and maximum severity was observed on Days 3 and 4 pc. The viral shedding rate was significantly lower in Group Mass/4-91 compared to other challenged groups. Serologic analysis in both experiments showed that the sera of challenged group exhibited significantly higher antibody titers than sera collected before challenge. Histopathologic investigations in SPF birds showed deciliation and hyperplasia in Group NV and less-pronounced lesions in Groups Mass/Mass and Mass. In commercial broilers vaccinated with H120 alone, hyperplasia and deciliation were observed in 90% of the tracheas. These experiments illustrated that Mor-It02 is pathogenic for chickens and a combination of live H120 and 4/91 vaccines given respectively at Day 1 and Day 15 of age confer a good protection against Mor-It02.

The emergence of enterococcal infections in neonatal broiler chickens in the poultry industry has become common in many countries, including Canada. The objective of this study was to examine the bacterial infections in nonviable broiler chicken embryos in three western Canadian poultry hatcheries using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The pattern of embryo mortality that occurred during incubation and the breakout analysis results were similar in all three hatcheries. The majority of embryo mortality occurred during the late stage of incubation (35.08%), followed by the early stage of incubation (15.35%). The breakout analysis showed that 65.82% of swabs had at least one type of bacterial growth while 34.17% of swabs were negative for bacterial isolation. Of those 65.82% swabs with bacterial growth, 34.3% of swabs yielded a mixed bacterial population while 31.52% yielded one type of bacterial growth. The frequency of bacterial isolation from hatch debris (60%–75%) increased with the age of broiler breeders. MALDI-TOF MS was able to provide genus-level identification of 83.13% of isolates among all bacterial types isolated. MALDI-TOF MS identified Enterococcus and Escherichia coli isolates with 97.18% and 100% accuracy at species level, respectively, whereas Staphylococcus species were identified with 62.59% accuracy. The congruence between MALDI-TOF MS identification and 16S rRNA or cpn60 universal gene target sequencing was 100% or 90%, respectively. Of all bacteria isolated, Enterococcus species (29.71%) were the most prevalent, followed by E. coli (19.46%). About 56% of E. coli–infected samples were coinfected with Enterococcus species. Among all Enterococcus species isolated, Enterococcus faecalis (79.58%) was the most prevalent, followed by Enterococcus faecium (8.1%). Overall, our study showed that Enterococcus-associated embryo mortality was predominant in all three hatcheries investigated and suggests that MALDI-TOF MS technology can be applied to identify bacteria such as Enterococcus species isolated from poultry.

Wooden breast (WB) is a fibrotic myopathy affecting the pectoralis major (p. major) muscle in fast-growing commercial broiler lines. Birds with WB are phenotypically detected by the palpation of a hard p. major muscle. A primary feature of WB is the fibrosis of muscle with the replacement of muscle fibers with extracellular matrix proteins, such as collagen. The ability of a tissue to be pliable and stretch is associated with the organization of collagen fibrils in the connective tissue areas surrounding muscle fiber bundles (perimysium) and around individual muscle fibers (endomysium). The objective of this study was to compare the structure and organization of fibrillar collagen by using transmission electron microscopy in two fast-growing broiler lines (Lines A and B) with incidence of WB to a slower growing broiler Line C with no phenotypically detectable WB. In Line A, the collagen fibrils were tightly packed in a parallel organization, whereas in Line B, the collagen fibrils were randomly aligned. Tightly packed collagen fibrils arranged in parallel are associated with nonpliable collagen that is highly cross-linked. This will lead to a phenotypically hard p. major muscle. In Line C, the fibrillar collagen was sparse in its distribution. Furthermore, the average collagen fibril diameter and banding D-period length were altered in Line A p. major muscles affected with WB. Taken together, these data are suggestive of different fibrotic myopathies beyond just what is classified as WB in fast-growing broiler lines.

Fowl cholera resulting from infection with Pasteurella multocida causes huge economic losses in the poultry industry. Necrotic hepatitis is reported to be a significant lesion associated with fowl cholera in chickens. Clarifying the underlying molecular mechanism of hepatic injury caused by P. multocida infection is needed to develop new strategies to control fowl cholera. Pasteurella multocida Q (the standard reference strain) and P. multocida 1G1 (a clinical strain) were used to infect healthy laying hens. Clinical signs were observed and gross lesions in livers were observed postmortem. Histologic lesions and the localization and expression of protein molecules associated with necroptosis, apoptosis, and inflammation in hepatic tissues were examined by hematoxylin and eosin staining and immunohistochemistry. Western blot analysis was used to determine the expression of liver injury–related genes. Necroptotic molecules such as RIPK1 (receptor interaction protein kinases 1), RIPK3 (receptor interaction protein kinases 3), and MLKL (mixed lineage kinase domain–like protein) were observed by immunostaining primarily in the cytoplasm of hepatocytes within or around necrotic foci, and inflammatory mediators HMGB1 (high-mobility group box 1) and IL-6 (interleukin-6) were found in the cytoplasm of heterophils, monocytes/macrophages, and hepatic sinusoids. In addition, MMP9 (matrix metalloproteinase 9) and TIMP1 (tissue inhibitor of metalloproteinase 1) were observed in hepatic parenchymal cells, inflammatory cells, and interstitial spaces, whereas the apoptotic effector molecule caspase-3 (cysteine-containing aspartic proteolytic enzymes 3) was mainly found in hepatocytes. The expression of RIPK1, RIPK3, and MLKL was significantly higher in the infected chickens than in the controls. HMGB1 and IL-6 protein levels were also increased in infected chickens relative to those in controls. Both MMP9 and TIMP1 were highly expressed in infected chickens. In addition, caspase-3 protein levels were significantly elevated in infected chickens. Necroptosis, apoptosis, and inflammation played a significant role in hepatic injury caused by P. multocida.

Synthetic oligodeoxynucleotides (ODN) containing unmethylated cytosine phosphodiester guanine (CpG) motifs (CpG-ODN) are effective immunostimulatory agents against a variety of viral, bacterial, and protozoan diseases in different animals including poultry. We have recently demonstrated that in ovo injection of CpG-ODN confers protection in neonatal chickens against bacterial septicemias. The objective of this study was to investigate the effectiveness of needle-free intrapulmonary (IPL) delivery of CpG-ODN microdroplets against Escherichia coli infection in neonatal chicks. In the present study, we used 880 chicks in total keeping 40 chicks per group. Chicks were delivered CpG-ODN or saline by IPL at the day 1 of hatch. Three days later, chicks were challenged with two doses (1 × 10⁴ CFU, n = 20 or 1 × 10⁵ CFU, n = 20) of E. coli. Chicks treated with CpG-ODN by the IPL route had significantly lower clinical signs and bacterial load compared to the group treated with saline (P < 0.05). CpG-ODN–treated groups were significantly protected against E. coli septicemia. We observed dose- and exposure time-dependent immunoprotective effects of IPL CpG-ODN in chicks. We found that IPL delivery of CpG-ODN can induce protective immunity as early as 6 hr that remains effective at least until day 5 post-treatment. Moreover, there were no adverse effects of IPL delivery of CpG-ODN on growth or mortality up to 42 days of age. Based on these findings, it can be suggested that CpG-ODN delivery by IPL route can be a promising alternative to antibiotics for inducing protective immunity in chicks during the critical first week of neonatal life.

Inclusion body hepatitis (IBH) is a disease affecting broiler chicken flocks worldwide. Several serotypes of fowl adenovirus (FAdV) have been implicated in disease outbreaks, with and without immunosuppression as a predisposing factor. IBH usually occurs in flocks up to 30 days of age; it is seldom seen in older birds. The objective of this study was to determine whether the pathogenicity for older birds of three FAdV field strains, belonging to serotypes 1, 8b, and 11, in the absence of immunosuppressive factors, was akin to that for younger birds, and to establish an effective and economical disease model for assessing cross-protection between serotypes. To achieve this objective, the gross pathology, histopathology, and dissemination of virus were examined at multiple time points after inoculation of 6-wk-old, specific-pathogen-free chickens via intraperitoneal injection. Both FAdV-8b and FAdV-11 generated lesions typical of those associated with outbreaks of IBH, and they were shown to be primary pathogens. The presence and severity of hepatic lesions were used to define two disease stages: degeneration (1–5 days postinoculation) and convalescence (6–14 days postinoculation). During the degenerative stage, FAdV-8b was detected in the liver, kidney, and gizzard of most birds, whereas FAdV-11 was predominantly detected in the liver, and both viruses persisted in the gizzard into convalescence. The pathogenesis of two IBH-associated FAdV strains in 6-wk-old chickens confirms their high level of virulence and also provides an effective experimental model for investigation of cross-protection between FAdVs. It also demonstrates persistence of the virus in the gizzard long after infection, supporting the notion that it is a site of viral shedding.

Avian influenza viruses (AIV) affect many species of birds including waterfowl and may persist in sediment in aquatic habitats. Sediment samples were collected from two areas representative of prime migration and overwintering waterfowl habitat in Dorchester County, Maryland in the fall and winter of 2013–2014. Samples were screened for the presence of AIV via reverse transcriptase–quantitative PCR targeting the matrix gene. Although 13.6% of sediment samples were positive for the AIV matrix gene across all collection dates and locations, differences in detection were noted with location and collection season. Percentage of AIV-positive sediment samples recovered corresponded to trends in waterfowl abundance at collection sites both temporally and spatially. These findings provide further support for the assertion that the presence of AIV in the aquatic environment is likely affected by the total number, site-specific density, and array of waterfowl species.

Helcococcus ovis is a slow-growing, pyridoxal-dependent, Gram-positive coccus belonging to the Peptostreptococcaceae family. Bacteria belonging to the genus Helcococcus are considered normal inhabitants of keratinized epithelium in humans; however, several reports support their role as pathogens in humans and several animal species. This case report describes the identification of H. ovis in a white leghorn rooster with valvular vegetative endocarditis and hepatitis. In February 2017 one dead, 7-yr-old, white leghorn rooster was submitted to the California Animal Health and Food Safety Turlock laboratory for diagnostic testing. Postmortem and microscopic examination revealed vegetative endocarditis and aortic thrombosis associated with large numbers of Gram-positive cocci. Myocarditis and extensive necrotic hepatitis were also noticed. Helcococcus ovis was isolated in large numbers from the aortic endothelium and confirmed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Bacterial colonies become evident 48 hr postincubation and exhibited a satellite growth around Escherichia coli on blood agar plates. A similar relationship has been described between Helcococcus spp. and Staphylococcus aureus. The primary site of infection in this chicken was not determined. To our understanding this is the first report of H. ovis infection in an avian species. The fastidious nature and nutritional requirements of Helcococcus spp. must be considered in order to allow proper identification and avoid misdiagnosis. Further studies are needed to define pathogenesis, virulence factors, and predisposing conditions associated with this microorganism.

Poxvirus was the causative agent of two unusual outbreaks of proliferative glossitis in canary (Serinus canaria forma domestica) breeders in the Northern Italy. A total of 45, 7–9-mo-old canaries were submitted in fair postmortem conditions to the Istituto Zooprofilattico Sperimentale delle Venezie at the beginning of November 2005 for diagnostic investigation. Birds belonged to two unrelated and geographically distant aviaries in northern Italy, herein identified as Aviary A and Aviary B. The two breeder flocks had both attended the same bird exposition held at the beginning of October and started experiencing an onset of high mortality 3 wk after the show. Twelve red factor-melanin canaries from Aviary A (Mantua) and 33 dominant white and recessive white canaries from Aviary B (Vicenza) were submitted for laboratory investigations. Clinical signs were unspecific and consisted of depression, ruffling of the feathers, epistaxis, and anorexia due to decreased feed and water intake. Postmortem findings revealed a severe increase in volume, thickening, and hardening of the tongue, which had turned pinkish to dark brown. No apparent gross lesions were noticed in integumentary, respiratory, and digestive systems or other internal organs. Histopathologic evaluation of the tongue revealed severe proliferation of the stratified squamous epithelium containing numerous large eosinophilic intracytoplasmic inclusion bodies (Bollinger bodies) displacing the nuclei of the cells peripherally. Severe ulceration of the surface epithelium, fibrinoheterophilic plaque formation, and moderate basal lymphoplasmacytic infiltrations were also associated with the proliferative lesion. Poxvirus was successfully isolated from the lesions in tissue cultures but not in specific-pathogen-free chicken embryonated eggs. Typical large, brick-shaped viral particles of 300–450 nm were also observed in affected tongues by transmission electron microscopy. This is the first report of multiple outbreaks of “poxvirus glossitis” in canaries.

Goose hemorrhagic polyomavirus (GHPV) is the causative agent of hemorrhagic nephritis enteritis of geese (HNEG), one of the major diseases of domestic geese in Europe. This case report describes a persistent outbreak of a GHPV infection on a Belgian goose farm. Clinical symptoms, necropsy lesions, and histopathologic lesions observed were compatible with previous reports of HNEG outbreaks. PCR analysis confirmed the diagnosis of GHPV. To our knowledge, this is the first report of an outbreak of a GHPV infection on a Belgian goose farm. This is evidence that GHPV is not only present in countries known for extensive waterfowl production, but disease outbreaks also occur in countries with less extensive goose production.