Anticoccidial drug resistance in coccidia has been known since the 1960s, and experimental selection for resistance in the laboratory has been done for most available products. However, routine testing for sensitivity in field isolates has only begun in recent years. Poultry producers are faced with greater challenges because of consumer preferences for ‘antibiotic-free’ products and a lack of new products for disease control. The classification of some of our most effective anticoccidials as ‘antibiotics’ has severely limited their use, causing us to rely on older products that are prone to resistance development. The interaction between coccidiosis and other diseases such as necrotic enteritis places more importance on routine testing for drug sensitivity. In this review, we have summarized the use of the anticoccidial sensitivity test (AST) as it is conducted and interpreted by poultry health specialists.

Bacillary white diarrhea in chickens was a major disease concern for the poultry industry during the early 1900s. Drs. L. F. Rettger and W. R. Hinshaw organized a meeting in 1928 to discuss methods for controlling this disease. In this meeting, representatives of five northeastern states discussed approaches to test for the presence of the etiological agent of bacillary white diarrhea, namely, Salmonella enterica subsp. enterica serovar Pullorum. Meeting attendees decided to have a yearly meeting of the Northeastern Conference of Laboratory Workers in Bacillary White Diarrhea. The next year, the name was changed to Conference of Laboratory Workers in Pullorum Disease Eradication, which was changed to Northeastern Conference on Avian Diseases (NECAD) in 1957. Not only has NECAD been important for the control of pullorum disease but also, starting with the fifth Annual Conference in 1932, other poultry diseases became an official part of the program. As such, NECAD served for a long time as the premier organization to present new information on avian diseases. The success of NECAD was based on the work of the many committees, which are described in detail in this review. For example, the antigen committee started officially in 1929 and remained active until at least 1987. The main task of this committee was to evaluate Salmonella Pullorum strains to be used by all participants in the pullorum antibody testing programs. NECAD started as a closed organization with participants from universities and government organizations but did not allow representatives from commercial groups until 1968 when all American Association of Avian Pathologists (AAAP) members in the Northeastern United States could participate. The journal Avian Diseases started with discussions by Dr. P. P. Levine with M. S. Cover, H. L. Chute, R. F. Gentry, E. Jungherr, and H. Van Roekel about the idea that NECAD would sponsor a journal dealing specifically with avian diseases. During the first few years of Avian Diseases publication, many articles including abstracts came from the NECAD Annual Conferences. The importance of NECAD as a precursor for other regional meetings and the AAAP and as a forum for graduate students to present their research are described. Several recipients of the award for the best paper presented by a graduate student have continued to work in avian disease research. The decline in the participation of scientists in the late 1990s and early 2000s was discussed extensively in 2006 and led to a merger of the NECAD meeting with the Pennsylvania Poultry Sales and Service Conference. Due to the COVID-19 pandemic, the 92nd Annual Conference was a virtual meeting in 2020. Fortunately, the 93rd Annual Conference in 2021 was an in-person meeting held in State College, PA.

Avian influenza viruses (AIVs) are distributed globally in members of the family Anatidae (waterfowl), and significant disease may occur when these viruses infect commercial poultry or humans. Early detection of AIV through surveillance of wild waterfowl is one measure to prevent future disease outbreaks. Surveillance efforts that are designed to account for host and environmental determinants of susceptibility to infection are likely to be most effective. However, these determinants have not been clearly delineated and may vary with location. Because some regions are at greater risk for AIV outbreaks, the factors that contribute to AIV infection of waterfowl in these areas are of interest. We investigated the prevalence of AIVs in hunter-killed waterfowl at wintering sites in California's Central Valley. Overall, AIV prevalence was 10.5% and, after controlling for age and sex, was greatest in northern shovelers (Spatula clypeata) and lowest in wood ducks (Aix sponsa). Overall, AIV prevalence was higher in females than in males, but this trend was driven by one sampling year and one waterfowl species (green-winged teal, Anas crecca). AIV prevalence in waterfowl was lower in samples collected from brackish wetlands compared with those collected from freshwater wetlands, suggesting that wetland type or other environmental factors contribute to AIV prevalence. This study adds to our understanding of the ecology of AIV infection in waterfowl and may assist in developing more efficient, targeted surveillance efforts for the detection of potentially harmful viruses circulating in North American waterfowl.

Avian influenza (AI) is a zoonotic disease significant to both public and animal health, caused by influenza virus A, and affects domestic poultry, wild birds, and mammals including humans. Aquatic birds are considered the natural reservoir of this virus. In 2016, Morocco experienced the first occurrence of low pathogenic H9N2 avian influenza virus (AIV) in poultry; however, no cases were reported in wild birds. The present study aimed to monitor the presence of AIV in wild birds in Morocco in order to trace the possible sources of the viruses affecting poultry. Between 2016 and 2019, 967 samples obtained from 480 birds representing 56 different wild bird species, 20 families, and 8 orders, mostly from Charadriiformes, Anseriformes, Pelecaniformes, and Passeriformes, were collected from various wetlands and relevant ornithologic sites in Morocco. These field samples consisted of 374 cloacal swabs, 321 tracheal swabs, 54 fecal samples, and 218 organ pools including the trachea, lung, liver, spleen, heart, intestine, and brain. The samples were examined for the presence of AIV using TaqMan-based real-time reverse transcriptase-PCR (rRT-PCR) targeting the matrix gene, followed by further subtyping rRT-PCR tests targeting the H1–H16 genes. The AI matrix gene was detected in 18 out of 967 samples (1.86%); positive samples were detected in 17 birds belonging to 10 bird species: two redshanks (Tringa totanus), one little stint (Calidris minuta), one ruddy turnstone (Arenaria interpres), one common snipe (Gallinago gallinago), one common greenshank (Tringa nebularia), one black-winged stilt (Himantopus himantopus), two black-headed gulls (Chroicocephalus ridibundus), one slender-billed gull (Chroicocephalus genei), six cattle egrets (Bubulcus ibis), and one Eurasian coot (Fulica atra). AIV was detected in 2 wetlands and 1 ornithologic site (Sidi Moussa Oualidia Complex, Smir lagoon and El Jadida Coast) and the highest positivity was revealed in fresh fecal samples (11.1%), indicating the suitability of this matrix for wild bird surveillance. Our results highlight that waders, gulls, and cattle egrets are the most affected species and may represent a potential risk for AI introduction in the poultry sector in Morocco. Regular monitoring of wild birds in Morocco, focusing in particular in the areas and species identified in this study as a high risk of virus circulation, should be implemented to anticipate and prevent possible AIV spread.

The intestinal disease coccidiosis, caused by parasitic Eimeria species, severely impacts poultry production, leading to an estimated $14 billion in annual losses worldwide. As the poultry industry moves away from antibiotics as a treatment for diseases, a better understanding of the microbiota is required to develop other solutions such as probiotics, prebiotics, and nutritional supplements. This study aimed to investigate the effects of Eimeria tenella infection on luminal (cecal contents [CeC]) and mucosal (cecal epithelial scrapings [CeS]) microbial populations in 288 Ross 708 broiler chickens at multiple time points postinfection (PI). By use of 16S rRNA amplicon sequencing, it was revealed that microbial diversity differed in infected (IF) chickens in comparison to the control (C) in both CeC and CeS microbiota at the peak of infection (7 days PI), when simultaneously IF birds saw reduced body weight gain and a higher feed conversion ratio. Infection resulted in a significant differential abundance of some bacterial taxa, including increases in potential secondary pathogens Escherichia coli, Enterococcus, Clostridium, and Proteus and a decrease in the short chain fatty acid-producing family Lachnospiraceae. Predicted metagenomic pathways associated with E. coli, such as those responsible for amino acid biosynthesis, were differentially expressed in IF birds. In conclusion, our results show that E. tenella infection disturbs luminal and mucosal microbiota balance in chickens. Moreover, the luminal microbiota seems to be more susceptible to prolonged imbalance due to IF, whereas the mucosal microbiota appeared to be affected only in the short term, demonstrating the importance of researching both the luminal and mucosal microbiota of the cecum.

Currently, there is no available vaccine against hemorrhagic enteritis virus (HEV) in Australia. Although it is assumed that subclinical HEV infections occur and may be associated with an increase in colibacillosis in Australian commercial turkey flocks, the prevalence of infection with this virus in the country is largely unknown. The aims of this study were to determine the extent of HEV infection in commercial flocks in Australia and to investigate the diversity of Australian HEV strains. Serum and spleen samples were collected from breeder and grower turkeys and serum was collected from breeder and grower chickens by the two major poultry integrator companies in Australia. Of the turkey samples, 727/849 (86%) sera were positive for anti-HEV antibodies by ELISA. HEV DNA was detected in 215/278 (77%) spleen samples positive by PCR. Of the meat chicken sera, 115/144 (80%) samples were seropositive. Sequencing the whole genome of three HEV field isolates showed that the Australian strains are highly similar and cluster separately from strains from other geographic regions although several point mutations were shared with HEV strains considered to be virulent. In conclusion, HEV infection is ubiquitous in Australian commercial poultry flocks. The impact of the many genomic point mutations detected in Australian HEV strains on virus pathogenicity is unclear.

Growing demand for poultry meat and eggs labeled as organic, cage free, or pasture raised has increased the number of producers that manage chickens outdoors. In these open environments, there are likely diverse enteric parasites sustained by fecal-oral transmission or passage through intermediate invertebrate hosts (e.g., worms and insects) that chickens consume. Enteric parasites can reduce chicken health and productivity, but there are few published data describing the identities or prevalence of these parasites on farms that use open environments in the United States. We surveyed 27 poultry farms with open environments that were situated across a wide geographic range, including California, Oregon, Idaho, and Washington. These farms did not use anticoccidial drugs, coccidia vaccines, or parasiticides. Flock size, enclosure area, flock density, flock rotation frequency, and average flock age were highly correlated for all the farms in this study. We analyzed how enclosure size and flock rotations per year (which represented two axes of variation in management) correlated with prevalence of five observed parasite taxa at the farm level. Across all flocks, we detected by fecal flotation Eimeria spp. (95% flocks), Ascaridia galli (69%), Heterakis gallinarum (52%), Capillaria spp. (39%), Strongyloides avium (13%), tapeworm species (29%), Cryptosporidium spp. (3%), and Dispharynx nasuta (1%). Eighty-five percent of samples were coinfected with two or more parasite taxa. Sixty-seven percent of farms raised only layer chicken breeds, 4% raised only broiler breeds, and 30% raised both layer and broiler breeds. The average age of the broiler flocks was 11.0 wk (±1.1 SE), and flocks were moved 54.7 (±17.9) times annually to new locations in pastures (hereafter, “rotation”). Layer flocks averaged 84.9 (±7.67) wk of age and were moved less often on farms being rotated 20.0 (±6.05) times per year. Generalized linear mixed models indicated that for every 1 m² increase in enclosure size, the odds of detecting Eimeria spp. increased by 0.03%. Furthermore, for every additional rotation per year, the odds of detecting A. galli decreased by 1.3%. For every additional rotation per year, the odds of detecting tapeworm species increased by 2.2%. We found no evidence that flock spatial management affected prevalence of the other parasites observed on the farms. Farming practices and parasite responses in these systems are highly varied, which makes it difficult to identify potential management interventions for reducing these infections.

The objectives of this study were to evaluate whether a preinfection of Eimeria adenoeides (EAD) or Eimeria tenella (ET) could affect the severity of subsequent histomoniasis in turkeys (Experiment 1) and if previous exposure to EAD infection, when a single or multiple inoculations of EAD were administered with sufficient time for complete cecal recovery, would affect the severity of HM incidence and lesions (Experiment 2). In Experiment 1, 200 poults were assigned to 1 of 5 groups, as follows: unchallenged negative control, positive challenge control inoculated with 10⁵ HM, EAD at 500 oocysts/bird and Histomonas meleagridis (HM), EAD at 2500 oocysts/bird and HM, or ET at 9 × 10⁶ oocysts/bird and HM. ET and EAD were inoculated on day 15 and HM on day 20. In Experiment 2, the trial consisted of two different challenge ages to evaluate short- or long-term EAD effects before HM challenge. Poults (n = 260) were assigned to either early-HM-challenged groups (HM on day 19 challenge control or EAD at 2500 oocysts/bird on day 14 with HM on day 19) or late-HM-challenged groups (HM on day 35 challenge control, EAD at 2500 oocysts/bird on day 14 and HM on day 35, or EAD at 100 oocysts/bird every 2–3 days during the first 3 weeks and HM on day 35). An unchallenged negative-control group was used for both the early- and late-challenge phases in Experiment 2. Mortalities were recorded, and surviving poults were scored for histomoniasis-related hepatic and cecal lesions. In Experiment 1, preinfection with both doses of EAD reduced the mortality as well as the cecal and hepatic lesions caused by histomoniasis. In Experiment 2, neither short- nor long-term preinfection with EAD had an effect on histomoniasis-related mortality or lesions. Differences between Experiments 1 and 2 may be due to the level of infection caused by the prechallenge with EAD and the resulting destruction of cecal tissue.

In 2018, a national recall of shell eggs in the United States occurred due to human illness caused by Salmonella Braenderup. Although previous studies have identified Salmonella Braenderup in laying hens and the production environment, little is known about the ability of this Salmonella serovar to infect laying hens and contaminate eggs. The objective of this study was to examine the invasiveness of Salmonella Braenderup in laying hens as well as its ability to persist in the production environment. Specific-pathogen-free laying hens (four trials; 72 hens/trial) were orally challenged with 10⁷ colony-forming units of Salmonella Braenderup. On day 6 postinoculation, half of the challenged hens were euthanatized, and samples of ileocecal junction (sections above and below it, and portions of both ceca), liver, spleen, ovary, and oviduct tissues were collected and cultured for Salmonella Braenderup. Egg and environmental (nest box swaps and substrate (litter)) samples were collected days 7–20 postinoculation (Trials 1 and 2; excluding weekends) and days 7–27 postinoculation (Trials 3 and 4; excluding weekends) to detect Salmonella Braenderup. Recovery of Salmonella Braenderup was highest in ileocecal tissue samples (11.1%–33.3%; P < 0.05), with little to no recovery in other collected tissue samples. Salmonella Braenderup was detected in a small number of shell emulsions (0%–2.9%; P < 0.01) and recovered in Trial 1 at a high rate (92.5%; P < 0.0001) in the substrate composite samples; however, recovery of Salmonella Braenderup was low in the other egg and environmental samples. These trials indicate that Salmonella Braenderup is not an invasive Salmonella serovar for cage-free laying hens, especially when compared to serovars of concern to the egg industry. However, it may persist in the environment at low levels.

Marek's disease (MD) vaccine does not provide sterilizing immunity that prevents subsequent MD virus (MDV) replication and shedding in vaccinated birds. It is hypothesized that cell-mediated immunity is critical to control the virus replication in chickens because MDV exists in cell-associated forms in the host. To improve the MD vaccine efficacy, particularly cell-mediated immunity, we constructed recombinant v301B/1-IL-15, an MDV serotype 2 vaccine strain 301B/1 expressing chicken interleukin-15 (IL-15), a cytokine which promotes T-cell proliferation and enhances T-cell responses. We examined the vaccine efficacy of v301B/1-IL-15 given as a bivalent MD vaccine in combination with turkey herpesvirus (HVT) against a very virulent MDV challenge. The expression of IL-15 did not interfere with virus stability and the growth of recombinant v301B/1-IL-15. However, the protective efficacy of v301B/1-IL-15 was not significantly different from that of v301B/1, the parental virus used to construct v301B/1-IL-15. Shedding of challenge virus was slightly reduced at Day 21 (16 days postchallenge) in the v301B/1-IL-15 plus HVT vaccinated group, with no statistically significant difference to that of the v301B/1 plus HVT vaccinated group, and thymus atrophy was observed to be less severe in the v301B/1-IL-15 plus HVT vaccinated group. Overall, the protection of v301B/1-IL-15 was not differentiable from v301B/1 against very virulent MDV challenge, but there is no interference with bivalent MD vaccine efficacy.

Runting stunting syndrome (RSS) in broiler chickens is characterized by altered intestinal morphology and gene expression and stunted growth. The objective of this study was to conduct a retrospective study of gene expression in stem and differentiated cells in the small intestine of RSS chicks. Two different models of RSS were analyzed: broiler chicks that were experimentally infected and broiler chicks that were naturally infected. Experimentally infected chicks were exposed to litter from infected flocks (RSS-litter chicks) or infected with astrovirus (RSS-astrovirus chicks). Intestinal samples from naturally infected chicks showing clinical signs of RSS were acquired from commercial farms in Georgia and were brought into a poultry diagnostic lab (RSS-clinical-GA) and from farms in Brazil that had a history of RSS (RSS-clinical-BR). The RSS-clinical-BR chicks were separated into those that were positive or negative for gallivirus based on DNA sequencing. Intestinal morphology and intestinal cell type were identified in archived formalin-fixed, paraffin-embedded tissues. In situ hybridization for cell-specific mRNA was used to identify intestinal stem cells expressing olfactomedin 4 (Olfm4), proliferating cells expressing Ki67, absorptive cells expressing sodium glucose cotransporter 1 (SGLT1) and peptide transporter 1 (PepT1), and goblet cells expressing mucin 2 (Muc2). RSS-litter and RSS-clinical-GA chicks showed 4% to 7.5% cystic crypts, while gallivirus-positive RSS-clinical-BR chicks showed 11.7% cystic crypts. RSS-astrovirus and gallivirus-negative RSS-clinical-BR chicks showed few cystic crypts. RSS-litter and gallivirus-positive RSS-clinical-BR chicks showed an increase in crypt depth compared to control or gallivirus-negative chicks, respectively. There was no expression of Olfm4 mRNA in the stem cells of RSS-litter and RSS-clinical-GA chicks, in contrast to the normal expression of Olfm4 mRNA in RSS-astrovirus and RSS-clinical-BR chicks. All chicks regardless of infection status showed normal expression of Ki67 mRNA in crypt cells, Muc2 mRNA in goblet cells, and SGLT1 or PepT1 mRNA in enterocytes. These results demonstrate that RSS, which can be induced by different etiologies, can show differences in the expression of the stem cell marker Olfm4.

Whole blood biochemistry and blood gas analysis are uncommonly used in poultry, but their use could improve the diagnosis of certain diseases or aid in monitoring flock health. To create preliminary reference intervals for selected blood analytes in broilers using the i-STAT and Vetscan VS2 (VS2) portable analyzers, we tested broilers at 7, 21, and 35 days of age. A total of 134 venous blood samples from healthy chickens of two different flocks were analyzed. There were significant age-related increases in concentration for glucose, hematocrit, ionized calcium, sodium, and carbon dioxide partial pressure on the i-STAT and for aspartate aminotransferase, creatine kinase, total calcium, phosphorus, and total protein on the VS2. Conversely, significant decreases in concentration were observed for pH, oxygen partial pressure, oxygen saturation on the i-STAT and for uric acid and albumin on the VS2. Additionally, significant differences were found on some blood parameters among the two flocks. Extremely high CK values were found on broilers after 21 days of age, indicating a possible degree of muscle injury during the grow-out. Preliminary reference intervals for all the analytes at each of the age groups were obtained. This study's data provide a starting point for the interpretation of blood analysis in broiler chickens at different ages and offer a new approach to investigate certain metabolic diseases that affect commercial poultry.

Resistance to infectious bronchitis (IB) is a polygenic trait, but little is known about how resistance distributes in the host population. In this study, a relatively large number (n = 369) of specific-pathogen-free white leghorn chickens (Gallus gallus) were challenged with an Arkansas -type virulent IB virus (IBV), and resistance was evaluated 5 days after challenge by viral load (IBV RNA) in the trachea and cecal tonsils, as well as by tracheal histomorphometry (mucosal thickness and lymphocyte infiltration). Contrary to expectations, results showed a non-Gaussian distribution of resistance of the whole population against challenge. Indeed, most chickens accumulated toward higher resistance, i.e., lower viral loads and less tracheal damage. The current results also indicated limited differences in resistance to IBV between sexes. Tracheal viral load was significantly higher in males than that in females, but tracheal damage did not significantly differ between sexes. The difference in tracheal viral load found in males and females could have implications for viral spread in commercial chicken populations.

In the last decade, monitoring Marek's disease (MD) vaccination by real-time PCR has become a common practice. Evaluating in vivo replication of MD vaccines in the feather pulp (FP) at 7 days of age provides information on how well a flock has been vaccinated. Factors such as vaccine dose, combination with other vaccines, age and route of vaccination, and the origin of the vaccine can influence the results and need to be taken into consideration. Early infection with oncogenic MD virus (MDV) could also affect how vaccines replicate in the first week and therefore might influence the results. The objective of this study was to evaluate if coinfection with oncogenic MDV could affect MD vaccine DNA viral load in the FP at 7 days of age. A retrospective study was done using data from nine animal experiments (46 treatment groups) in which chickens were vaccinated against MD either in ovo or at 1 day of age and challenged with various oncogenic strains at 1 day of age by contact. In each experiment, vaccinated but not challenged groups were used as controls. Replication of MD vaccine was evaluated in samples of FP collected at 7 days of age by real-time PCR, and percentage of positives and vaccine load were analyzed. Our results show that CVI-988 (13 treatment groups), SB-1 (six treatment groups), and in most cases turkey herpesvirus (HVT; 24 out of 27 treatment groups) replication was not affected by early infection with oncogenic MDV. There were three treatment groups in which HVT replication differed between challenged and unchallenged chickens, however the effect was not clear; replication of HVT in nonchallenged chickens was higher (one treatment group) or lower (two treatment groups) than in challenged chickens and factors other than coinfection with MDV might have contributed to such differences.

Infectious bronchitis is a respiratory disease of chickens caused by a gammacoronavirus named infectious bronchitis virus (IBV). In addition to affecting the respiratory tract, IBV may also induce urogenital infections, leading to nephropathogenic disease, false layer syndrome in laying hens, and epididymal lithiasis and epididymitis in males. Here, we report a case of decreased reproductive efficiency due to male infertility in 33- to 38-wk-old broiler breeders. At necropsy, the males presented with urates deposited on the skin around the vent and testicular asymmetry due to marked unilateral atrophy. Histopathology revealed lymphocytic epididymitis, epididymal lithiasis, and orchitis. IBV antigen was detected within collecting and efferent ducts of epididymides by immunohistochemistry. IBV strain DMV/1639 was detected by reverse transcription-quantitative PCR in pools of testes, oviducts, tracheas, cecal tonsils, and kidneys from a 37-wk-old affected flock. This report shows evidence of the role of IBV in male chicken infertility and highlights the importance of performing molecular surveillance of IBV to monitor vaccine strains and to detect emerging variants that can potentially hinder production.

Fowl glioma-inducing virus (FGV), a strain of avian leukosis virus (ALV) subgroup A, is the causal agent of fowl glioma characterized by multiple nodular astrocytic growths, gliosis, and lymphocytic encephalitis. Also associated with FGV infection are cases of cerebellar hypoplasia, perineuromas, and nonsuppurative myocarditis. Though fowl glioma has been recognized in several countries, most reports of FGV infection come from Japan. A 9-mo-old brown leghorn from a German farm with nine leghorns was presented to a veterinarian with an impaired general health with torticollis, tremor, and incoordination. Histopathology revealed multifocal nodular astrocytic growths, gliosis, and a lymphoplasmacytic encephalitis. Immunohistochemically, neoplastic astrocytes showed positivity for anti-ALV antibody. FGV was detected in the brain with nested reverse transcription-polymerase chain reaction (RT-PCR) and subsequent sequencing of PCR product. The remaining eight birds were screened for the presence of ALV with real-time RT-PCR. Four leghorns tested positive for exogenous ALV in nested RT-PCR with an identical nucleotide sequence as the leghorn with neurological symptoms. To the authors' knowledge this is the first report of a natural FGV infection in a brown leghorn in Germany with clinical manifestation.

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