One dead 6-wk-old male racing pigeon (Columba livia) was submitted for postmortem evaluation after presenting with weight loss, anorexia, dry shanks, dehydration, and lethargy. The bird belonged to a confined flock with 12 other pigeons raised by a hobbyist. Two pigeons in the flock reportedly had died with a history of similar clinical signs. On gross examination, the liver and the spleen were diffusely dark brown to black. Histopathology revealed moderate to large amounts of anisotropic, intracytoplasmic black pigment, compatible with hemozoin, in the spleen, liver, lung, and kidneys, with small amounts in the heart and meninges of the brain. Marked plasmacytic infiltrates were observed in liver, lungs, heart, and kidneys. Blood smears from a clinically affected concomitant pigeon from the flock revealed numerous light-blue, round to oval, intraerythrocytic trophozoites and meronts suggestive of Plasmodium spp. PCR and sequencing tests were performed from spleen and ceca with fragments of the 18S ribosomal RNA and the mitochondrial cytochrome b (cytB) genes. Sequencing results confirmed the presence of Plasmodium in the affected pigeon. Although an exact genetic match could not be determined, the most similar species to the isolate from this study are Plasmodium relictum, Plasmodium matutinum, Plasmodium lutzi, and Plasmodium homocircumflexum.

Movement and land application of manure is a known risk factor for secondary spread of avian influenza viruses. During an outbreak of highly pathogenic avian influenza (HPAI), movement of untreated (i.e., fresh) manure from premises known to be infected is prohibited. However, moving manure from apparently healthy (i.e., clinically normal) flocks may be critical, because some egg-layer facilities have limited on-site storage capacity. The objective of this analysis was to evaluate targeted dead-bird active surveillance real-time reverse transcriptase polymerase chain reaction (rRT-PCR) testing protocols that could be used for the managed movement of manure from apparently healthy egg-layer flocks located in an HPAI control area. We also evaluated sequestration, which is the removal of manure from any contact with chickens, or with manure from other flocks, for a period of time, while the flock of origin is actively monitored for the presence of HPAI virus. We used stochastic simulation models to predict the chances of moving a load of contaminated manure, and the quantity of HPAI virus in an 8 metric ton (8000 kg) load of manure moved, before HPAI infection could be detected in the flock. We show that the likelihood of moving contaminated manure decreases as the length of the sequestration period increases from 3 to 10 days (e.g., for a typical contact rate, with a sample pool size of 11 swabs, the likelihood decreased from 48% to <1%). The total quantity of feces from HPAI-infectious birds in a manure load moved also decreases. Results also indicate that active surveillance protocols using 11 swabs per pool result in a lower likelihood of moving contaminated manure relative to protocols using five swabs per pool. Simulation model results from this study are useful to inform further risk evaluation of HPAI spread through pathways associated with manure movement and further evaluation of biosecurity measures intended to reduce those risks.

The history of pullorum disease is closely intertwined with the history of avian health research and that of the poultry industry. The seriousness of the disease galvanized the attention and brought together, for the first time, the pioneers of poultry health research to work cooperatively on different aspects of the disease. Control of the disease made it possible for intensive poultry production to develop as the basis for the modern poultry industry. During the early 1900s, bacillary white diarrhea (BWD) was a devastating disease of young chickens threatening the developing poultry industry. Dr. Leo F. Rettger isolated and described the bacterial pathogen, Salmonella enterica serotype Pullorum, for the first time in 1900. BWD was renamed pullorum disease in 1929. In subsequent years, Rettger and coworkers were able to reproduce the disease and fulfill Koch's postulates. Rettger et al. also showed that Salmonella Pullorum was vertically transmitted, which was the first time that a pathogen was shown to be vertically transmitted. The development of serologic tests was of crucial importance because it led to the development of effective eradication methods to identify carrier birds and to exclude these birds from the breeder flocks. The negative impact of pullorum disease on the poultry industry ultimately was one of the major reasons that the National Poultry Improvement Plan (NPIP) was developed by scientists, the poultry industry, and the United States Department of Agriculture (USDA). Needless to say, the work of the pioneering researchers formed the basis for the control of the disease. The NPIP started in 1935, with 34 states participating in testing 4 million birds representing 58.2% of the birds hatched. The program rapidly expanded to 47 states by 1948 and tested more than 30 million birds. In 1967, all commercial chicken hatcheries participating in the NPIP were 100% free of pullorum and typhoid disease caused by Salmonella enterica serotype Gallinarum. This historical overview of pullorum disease describes in some detail the progress made, especially during the early years, toward controlling this disease using methodologies that were often very basic but nonetheless effective. One has to admire the ingenuity and persistence of the early researchers leading to their achievements considering the research tools that were available at the time.

Histomonas meleagridis is a trichomonad protozoan parasite that can cause an important poultry disease known as histomoniasis; Marek's disease virus (MDV) and subtype J avian leukosis virus (ALV-J) usually cause avian oncogenic diseases. Although these diseases have been reported in a single pathogen infection, information about their coinfection is scarce. This study reports a naturally occurring case of coinfection with H. meleagridis, MDV, and ALV-J in a local chicken flock at the age of 150 days. Necropsy revealed necrosis and swelling in the liver and spleen. Histologic analysis showed large areas of mild to severe necrosis of hepatocytes, with numerous intralesional trophozoites of H. meleagridis by H&E and periodic acid-Schiff staining; H&E staining showed pleomorphic and neoplastic lymphoid tumor cells in the liver and myeloid cells with eosinophilic cytoplasmic granules in the spleen. Coexpression of MDV and ALV-J antigens was detected in the liver by fluorescence multiplex immunohistochemistry staining. The 18S rRNA gene of H. meleagridis, meq gene of MDV, and gp85 gene of ALV-J were identified in mixed liver and spleen tissues by PCR and sequencing, respectively.

Marek's disease virus (MDV) is an important poultry pathogen that is controlled through widespread vaccination with avirulent and attenuated strains. However, continued evolution of field viruses to higher virulence has required ongoing improvement of available vaccine strains, and these vaccine strains offer an attractive platform for designing recombinant vector vaccines with cross-protection against MDV and additional pathogens. Recent reports of failures in vaccine licensing trials of positive controls to reach appropriately high levels of Marek's disease incidence prompted us to evaluate possible combinations of outbred specific-pathogen-free layer lines and alternative virulent challenge strains that could provide more consistent models for serotype 3 vectored vaccine development. Choice of layer line and virulent MDV challenge strain each contributed to the ability of a challenge model to reach 80% virulence in unvaccinated positive control groups in the majority of trials, without overwhelming serotype 3 vectored vaccine protection in vaccinated groups. Conversely, reducing challenge virus dose by a factor of four, or vaccine dose by half, had no consistent effect across these models. Although MDV strain 617A had the most potential as an alternative to strains that are currently approved for licensing trials, no combination of layer line and challenge virus consistently met the goals for a successful challenge model in all study replicates, indicating that high variability is an inherent difficulty in MDV challenge studies, at least when outbred birds are used.

The aim of this study was to evaluate and quantify the parasitological challenge in pastured poultry production in the state of Georgia. Over the course of 1 yr, fecal samples from six turkey flocks, 10 broiler flocks, and 13 layer flocks were collected on a pastured farm in 2-wk intervals to determine counts of Eimeria oocysts and nematode eggs. Average coccidia counts were 10,198 oocysts per gram of feces (OPG) in broiler flocks, 1470 OPG in layer flocks, and 695 OPG in turkey flocks. The means in broiler and turkey flocks were higher at their first week on pasture. Counts in broilers and layers were significantly higher in spring than in winter and summer. Coccidia counts in broilers were lower than published numbers in conventionally reared poultry, indicating the rotation system of the pastures might effectively reduce the infection pressure. Next-generation sequencing of PCR products showed the presence of most described Eimeria spp. in broilers, layers, and turkeys. In addition, operational taxonomic units (OTUs) x, y, and z were found. The frequency of species was similar for broilers and layers, with the exception that Eimeria praecox and OTU z were more common in layers. In layer flocks, the average count of roundworm eggs per gram of feces (EPG) was 509 EPG with 80% of the samples being positive. The mean counts had no clear pattern related to age. There was an increase of EPG with the increase of temperatures during spring and summer with the peak at midfall. Worm eggs from laying hens were identified as Ascaridia galli. The seasonal differences suggest that higher temperatures might result in an increase of egg survival and sporulation in the environment.

A multiage commercial layer pullet operation with a history of chicken embryo–origin (CEO) modified live infectious laryngotracheitis (ILT) virus vaccination suffered severe ILT outbreaks in 2017. The initial sequencing revealed that the circulating virus was of vaccine origin. Changes to the timing and dosage of CEO ILT vaccine failed to control the outbreak. The clinical resolution of the outbreak occurred with the transition to a turkey herpesvirus vector vaccine given in-hatchery, followed by a tissue culture–origin vaccine given on the farm. The circulating ILT viruses were monitored periodically by next-generation sequencing. This site became free of ILT virus within 1 yr after implementing the new vaccination program.

The resistance to serum complement-mediated killing is a vital virulence property of microbial pathogens. Complement factor H (FH) is a key negative regulator of the complement alternative pathway (AP) that prevents formation and accelerates the decay of AP C3 convertase and acts as a cofactor in the inactivation of C3b. Pathogens can recruit host FH through their surface proteins to escape the clearance of the complement system. Riemerella anatipestifer could also evade the complement system attack to achieve host infection, but the mechanism is still unclear. In this study, the R. anatipestifer proteins that could interact with FH in host serum were screened and analyzed, and the functions were determined. Affinity chromatography with a Ni–nitrilotriacetic acid Sefinose column and mass spectrometry identified three outer membrane proteins (Omp) of R. anatipestifer, Omp54, Omp53, and Omp24, as potential FH-binding proteins. We then successfully conducted the prokaryotic expression and polyclonal antibody preparation of three candidate proteins. Indirect immunofluorescence assay showed that three candidate proteins were all present in R. anatipestifer. The affinity blotting assay, anti-serum–inhibiting assay, and serum bactericidal assay presented evidence that Omp24 could bind FH. Moreover, FH bound to Omp24 was associated with resistance to the alternative pathway and functional for R. anatipestifer survival in the normal duck serum. These results suggested that R. anatipestifer Omp24 was a FH-binding protein and the interaction with FH blocked the alternative pathway. Recruitment of complement regulatory proteins may facilitate better R. anatipestifer resistance to this vital line of host defense.

Hemorrhagic hepatopathy is a syndrome reported in layer pullets resulting in mortality and lesions including hepatic, splenic, and intestinal necrosis; hepatic and splenic enlargement; hemorrhages; amyloidosis of the muscle, spleen, and liver; accumulation of noncoagulated hemorrhagic fluid in the coelom; and frequently, granulomatous myositis at bacterin injection sites. The syndrome is characterized in the literature in table egg layer pullets and is thought to be associated with the administration of bacterin vaccines, namely, frequently Salmonella enterica subsp. enterica bacterins. Hemorrhagic hepatopathy is recognized by industry veterinarians as also occurring infrequently in broiler breeder pullets in the United States. As the condition is likely due to an inflammatory process in response to bacterial lipopolysaccharide inoculation, it is important to characterize both the pathologic changes and predisposing factors for the condition in broiler breeds, which are immunologically different from table egg layer breeds. In this study, we characterize the gross and microscopic lesions observed in a series of diagnostic laboratory cases of hemorrhagic hepatopathy in broiler breeder pullets and suggest a possible pathophysiology for the condition. Additionally, we report results from a case survey of the United States broiler industry that suggest that the condition is due to a reaction to bacterin vaccination and that certain bacterin products may predispose pullet flocks to develop the condition. Although further research is indicated, these findings establish hemorrhagic hepatopathy as a pathologic condition of broiler breeder pullets and may aid in the diagnosis and prevention of the syndrome.

Duck viral hepatitis (DVH) mainly affects ducklings under 1 month of age, causes liver necrosis, enlargement, and hemorrhage, and is highly lethal, seriously jeopardizing the duck industry. The prevalence of duck hepatitis A virus (DHAV-1) and duck astrovirus type 3 (DAstV-3) is increasing, and coinfection is common. Moreover, the similar clinical characteristics of the DHAV-1 and DAstV-3 infections and the high frequency of coinfection make diagnosis difficult. In this study, to establish a method for the rapid, simultaneous detection of DHAV-1 and DAstV-3, two pairs of specific primers were designed according to their conserved gene regions. An SYBR^® Green I-based qPCR assay was successfully established that can quickly and differentially detect the two viruses. Moreover, the assay is highly specific and does not show cross-reaction with other common viruses. The detection limit of the method is 7.34 × 10¹ copies/µl and 3.78 × 10¹ copies/µl for DHAV-1 and DAstV-3, respectively, indicating high sensitivity. A total of 34 clinical samples were tested using the established method; the positive rates for DHAV-1 and DAstV-3 were 14.71% and 8.82%, respectively, and that for coinfection was 2.94% (1/34), which was better than that obtained with conventional PCR. In summary, the SYBR Green I-based qPCR assay established in this study has high specificity, good sensitivity and accuracy, high feasibility, and is rapid. Thus, it can be a powerful tool for the coinfection detection of DHAV-1 and DAstV-3 and for future epidemiologic studies.

As part of a 2 yr disease surveillance project of small poultry flocks, owners of birds submitted for postmortem examination to the Animal Health Laboratory were asked to complete a questionnaire designed to gather information on the characteristics of the flock and its environment, how the flock was managed, and biosecurity measures used. A total of 153 unique questionnaires were received. Personal consumption of meat or eggs was the most common reason for owning a small flock (69.3%). Almost all owners (97.4%) reported having chickens on their property, while 21.6% had waterfowl, 15.7% had turkeys, and 15.7% had game birds. Nearly 70% (69.9%) of the flocks had some degree of outdoor access. For those with indoor access, the most common bedding material provided was soft wood shavings (70.2%). Kitchen waste or leftovers were offered to 65.3% of flocks, and well water was the most common source of drinking water (80.6%). For flocks with indoor access, dedicated shoes and clothes were used when entering or cleaning the coop by fewer than half of owners, and shoes were rarely disinfected before or after contact with the flock. Most owners (93.8%) reported washing their hands after contact with their birds, although only 48.3% reported washing their hands before contact. Among owners who sourced birds from a hatchery, only 36.8% indicated that the birds had been vaccinated, and 21.1% were unsure if vaccines had been administered. Among owners using medication (60.5%), the use of antibiotics was common (60.9%). Overall, questionnaire responses describe a wide range of husbandry and biosecurity practices, often suboptimal, and point out the need for educational material for Ontario small flock owners.

Several serotypes of non-host-specific or paratyphoid Salmonella have been linked with contamination of poultry meat, and eggs, resulting in foodborne outbreaks in humans. Vaccination of poultry against paratyphoid Salmonella is a frequent strategy used to reduce the levels of infection and transmission, which ultimately can lead to lower rates of human infections. Live vaccines have been developed and used in poultry immediately after hatching as a result of their ability to colonize the gut, stimulate a mucosal immune response, induce a competitive inhibitory effect against homologous wild strains, and reduce colonization and excretion of Salmonella. Furthermore, vaccines can competitively exclude some heterologous strains of Salmonella from colonizing the gastrointestinal tract when young poultry are immunologically immature. In addition, various studies have suggested that booster vaccination with live vaccines a few weeks after initial vaccination is essential to increase the level of protection and achieve better cross-protective immunity. Vaccination of breeders, broilers, layers, and turkeys with modified live Salmonella vaccines is a common intervention that has become an important component in poultry companies' multistep prevention programs to meet increasingly demanding customer and regulatory food safety requirements. Both live and inactivated vaccines play a critical role in a comprehensive control program for chicken and turkey breeders and commercial layers. This review examines the response and protection conferred by live modified vaccines against non-host-specific Salmonella that can be considered for the design and implementation of vaccination strategies in poultry.

Artificial insemination is a routine practice for turkeys that can introduce pathogens into breeder flocks in a variety of ways. In this manuscript, a risk analysis on the potential transmission of highly pathogenic avian influenza (HPAI) to naïve hens through artificial insemination is presented. A case of HPAI on a stud farm where the potential transmission of the virus to susceptible hens in the 2015 H5N2 HPAI outbreak in Minnesota is described along with documentation of known and potential transmission pathways from the case. The pathways by which artificial insemination might result in the spread of HPAI to susceptible hens were determined by considering which could result in the 1) entry of HPAI virus onto a premises through semen movement; and 2) exposure of susceptible hens to HPAI as a result of this movement. In the reported case, HPAI virus was detected in semen from infected toms, however, transmission of HPAI to naïve hens through semen is unclear since the in utero infectious dose is not known. This means that the early detection of infection might limit but not eliminate the risk of hen exposure. Because of the numerous potential pathways of spread and the close contact with the birds, it is highly likely that if semen from an HPAI-infected tom flock is used, there will be spread of the virus to naïve hens through insemination. If insemination occurs with semen from stud farms in an HPAI control area, receiving hen farms should have restricted movements to prevent outbreak spread in the event that they become infected.

In April and November of 2018, multiple commercial laying hen flocks within the same company presented with a sharp increase in mortality and drop in egg production that persisted for several days. These flocks showed striking necropsy lesions consistent with systemic infection and responded to antimicrobial treatment in the feed. Staphylococcus aureus (SA) was the most frequently isolated organism from multiple tissues including comb and wattle lesions, lungs, liver, ovary, spleen, and bone marrow. Given such an uncommon presentation of SA, which is known as a secondary opportunistic pathogen, a challenge study was conducted to evaluate its role in these disease outbreaks. In the present study, laying hens of two ages (22 and 96 wk) were inoculated with SA via three routes: oral gavage, subcutaneous (SC) injection, and intravenous (IV) injection. Both young and old hens in the IV group showed a significant increase in body temperature and drop in body weight; however, the clinical signs observed in the naturally occurring outbreaks were not present. SA was reisolated at multiple time points postchallenge from all challenge groups except the negative control group. While the SC group showed localized necrosis at the injection site, microscopic changes were different from changes observed in birds from the natural outbreaks. Despite observed initial differences in route and age, the SA challenge strain was not capable of reproducing the disease on its own. The results of this study indicate that SA may have played a role in the increased mortality, clinical signs, and necropsy lesions reported with the naturally occurring outbreaks. However, SA should still be considered as a secondary opportunistic pathogen. Other factors that could have caused the initial insult are stress, immunosuppression, or other primary infectious agents. The results of this study may aid veterinary diagnosticians, clinicians, and all poultry professionals to include SA in their differentials list as a secondary opportunistic pathogen in similar cases. This is an uncommon presentation and further field observations and clinical studies are needed to better elucidate the pathogenesis of this disease, which will in turn help to prevent future outbreaks.