Genetic characterization of Marek's disease virus in chickens in Thailand reveals a high genetic diversity of circulating strains.

Marek's disease (MD) is a highly contagious lymphoproliferative disease of chickens caused by Gallid alphaherpesvirus 2, commonly known as serotype 1 Marek's disease virus (MDV-1). Despite widespread vaccination, MD-related cases have been frequently observed worldwide, including in Thailand. However, no information is available on the genetic characteristics of MDV-1 field strains circulating in chickens in Thailand. This study investigated the geographic distribution and genetic characteristics of MDV-1 field strains circulating in chickens in Thailand between 2013 and 2021 by analysing the Meq and pp38 genes. Out of a total of the 286 clinical samples obtained from 70 chicken farms located in major chicken raising areas of Thailand, 138 samples (48.25%) from 46 chicken farms (65.71%) tested positive for MDV-1 field strains. Results demonstrated that MDV-1 field strains were extensively distributed in major chicken raising areas. Phylogenetic analyses based on the Meq gene revealed that four clusters of MDV-1 circulated in chickens in Thailand between 2013 and 2021. Among these clusters, cluster 1 was the predominant cluster circulating in chickens in Thailand. Additionally, our findings based on molecular characteristics of Meq and pp38 gene/protein suggested that most of the Thai MDV-1 field strains were potentially highly virulent. In conclusion, our data demonstrated the circulation of different clusters of MDV-1 with virulence characteristics in chickens in Thailand, indicating high genetic diversity of MDV-1 in Thailand. This study highlights the importance of more effective vaccine development and routine MDV-1 surveillance for early detection and control of highly virulent MDV-1.

The reversible effects of gossypol toxicity on male pigeons' reproductive performance.

Background and Aim: Gossypol, a cotton seed derivative, is well known for its reversible antifertility in male reproduction across species. Its antifertility and reversibility effects on male reproductive function vary among species in dose-and time-dependent manners. In this study, the antifertility potential of gossypol in pigeons was evaluated for the first time to determine whether it might be used as a dietary supplement for pigeon population control. Materials and Methods: Male pigeons were assigned into three experimental groups: The gossypol-treated group (n = 12), the sham control group (n = 6), and the negative control group (n = 6). There were two experimental periods: A gossypol-feeding period of 28 days and a gossypol-free period of 28 days. During the gossypol-feeding period, birds in the gossypol-treated group were fed 4 mg of gossypol extract per day. Birds in the sham control group were fed 0.5 mL of mixed ethanol and sunflower oil, while those in the negative control group were fed 0.5 mL of phosphate buffer saline. After the gossypol-feeding phase was completed, all remaining pigeons in all groups continued to receive their regular diet for an additional 28 days (gossypol-free phase). The body weight and semen quality of the birds in the experimental groups were compared to evaluate gossypol's antifertility effect. Results: In the gossypol-treated group as compared to the control groups, the percentages of sperm motility and viability were significantly lower at 21 days, and the percentage of normal sperm morphology was significantly lower at 28 days during the gossypol-feeding period. After gossypol withdrawal, these antifertility effects were resumed and reached a comparable semen quality to the control groups within 14 days. Conclusion: Gossypol supplementation (4 mg/day for 28 days) could lower male pigeons' reproductive performance in terms of sperm motility, viability, and sperm morphology. Such infertility was, however, reversible within 14 days after gossypol withdrawal without any side effects on the pigeons, suggesting its application as a safe contraceptive feeding for male pigeons.

Seasonal effect on semen availability and quality of racing pigeon in Thailand.

BACKGROUND AND AIM: Seasonal variations among geographical regions could influence pigeon semen quality differently. This study aimed to determine the seasonal effect on semen availability and quality of racing pigeons in Thailand to understand and improve breeding management in the country. MATERIALS AND METHODS: Semen was collected from six fertile captive pigeons once a week during summer (March-June), monsoon (July-October), and winter (November-February) during 2019-2020. The success rate of semen collection and semen quality was determined in each season - by which changes in average temperature, humidity, and photoperiod were obtained. RESULTS: Comparable success rates of semen collection were acquired among different seasons, while varied semen qualities were revealed. The percentages of total motility and progressive motility score of sperm were significantly lowest in summer (66.35±3.40 and 3.88±0.15, respectively) compared to monsoon (85.45±2.91 and 4.67±0.10, respectively) and winter (79.29±1.96 and 4.37±0.10, respectively), while its concentration (×109 sperm/mL) and outputs (×106 sperm) were significantly highest in winter (7.62±0.54 and 91.44±10.83, respectively) compared to summer (4.23±0.41 and 48.45±6.35, respectively) and monsoon (3.57±0.30 and 51.45±7.21, respectively). Besides, semen samples collected from birds housing at an average temperature of <29.5°C demonstrated better sperm motility sperm concentration and total sperm counts than those from at a higher temperature. CONCLUSION: Winter was regarded as the best season contributing the best semen quality, while summer was the worst. Due to the fluctuation of temperature during summer and winter, the seasonal temperature was implied as the major factor contributing to changes in sperm quality of racing pigeons in Thailand.

Comparison of three filter paper-based devices for safety and stability of viral sample collection in poultry.

General diagnosis of poultry viruses primarily relies on detection of viruses in samples, but many farms are located in remote areas requiring logistic transportation. Filter paper cards are a useful technology that offer an alternative for collecting and preserving samples without hazardous exposure. The goal of this study was to compare three filter papers: the Flinders Technology Associates filter (FTA®) card, dried blood spot (DBS) card and qualitative filter paper (FP) grade 2 to collect poultry samples. In particular, we have used Newcastle disease virus (NDV) to evaluate safety and a Marek's disease virus (MDV) attenuated vaccine (CVI988) to evaluate stability of viral DNA. This experiment was divided into two parts. The first part was to determine the DNA stability and detection limit of CVI988 in samples collected in different paper supports after four storage times (3, 7, 14 and 30 days post spot). The second part was to determine the safety of papers by evaluating the viral inactivation efficacy using NDV as a representative virus. Results showed that all papers could preserve CVI988 DNA at all times, with a detection limit of 0.5 PFU/5 µl for FTA® and DBS cards, and 5 PFU/5 µl for FP. Our results showed that the NDV remained viable and infectious on the DBS card and FP, while no viable virus was detected on the FTA® card, suggesting that the FTA® card was safest to use. Therefore, the use of the DBS card and FP for infectious sample collection should be discouraged and reconsidered. RESEARCH HIGHLIGHTS The detection limits of the FTA® card, DBS card and FP for CVI988 detection were 0.5, 0.5 and 5 PFU/5 µl, respectively. All three filter papers could preserve viral DNA for at least 30 days of post spot. The DBS card and FP are not suitable for collecting NDV samples, which is one of the major economical threats for the poultry industry worldwide.

Genetic characterization of reticuloendotheliosis virus in chickens in Thailand.

Reticuloendotheliosis virus (REV) causes an immunosuppressive, runting, and oncogenic disease in poultry, posing a significant threat to the poultry industry. In Thailand, an unidentified disease associated with runting-stunting syndrome and neoplasia, resembling REV infection, has been continuously observed in several chicken farms. However, REV infection in Thailand has never been reported. In this study, we investigated the occurrence and genetic characteristics of REVs in chickens in Thailand from 2013 to 2016. Of the 130 clinical samples obtained from 29 chicken farms from 9 provinces located in the major chicken-raising regions of Thailand, including the central, eastern, northern, and northeastern parts of Thailand, 51 samples (39.23%) and 21 farms (72.41%) were REV-positive. REV-positive samples were detected in all 9 provinces tested. Our results demonstrated that REV was extensively distributed in the major chicken-raising regions of Thailand. Phylogenetic analysis of the whole genome sequence showed that Thai REV was most closely related to Chinese, Taiwanese, and the US REV strains isolated from different avian species and clustered into REV subtype III. This finding indicates that REV subtype III was predominantly circulated in Thai chicken flocks. This study is the first report on REV infection in chickens in Thailand. Our findings raise the awareness of REV as another causative agent of runting and oncogenic disease in chickens in Thailand and highlight the wide distribution of REV infection among chickens worldwide.

Persistence of Chlamydia psittaci in Various Temperatures and Times.

Chlamydia psittaci, an obligate intracellular gram-negative bacteria, causes an important zoonotic disease in humans, namely, psittacosis. The objective of this study was to determine the persistent viability of C. psittaci at various temperature conditions. The cloacal swab samples were collected from feral and racing pigeons to find a C. psittaci field strain. The bacterial isolation showed that 1.3% of feral pigeons were PCR positive, while all samples of racing pigeons were PCR negative. Also, bacterial characterization suggested that it belonged to genotype B, which had bacterial titers 3.2 and 3.89 log 50% lethal dose/ml, respectively. A bacterial persistence test was performed, and the results showed that C. psittaci could survive at 56 C for up to 72 hr. In conclusion, C. psittaci could be found in feral pigeons in central Thailand. The bacteria can survive in equatorial temperature areas. This study was the first to report that C. psittaci could survive and has infectivity at 56 C for 72 hr. Therefore, awareness of C. psittaci infection in humans is necessary and should be a public health concern.

Comparative study of pandemic (H1N1) 2009, swine H1N1, and avian H3N2 influenza viral infections in quails.

Quail has been proposed to be an intermediate host of influenza A viruses. However, information on the susceptibility and pathogenicity of pandemic H1N1 2009 (pH1N1) and swine influenza viruses in quails is limited. In this study, the pathogenicity, virus shedding, and transmission characteristics of pH1N1, swine H1N1 (swH1N1), and avian H3N2 (dkH3N2) influenza viruses in quails was examined. Three groups of 15 quails were inoculated with each virus and evaluated for clinical signs, virus shedding and transmission, pathological changes, and serological responses. None of the 75 inoculated (n = 45), contact exposed (n = 15), or negative control (n = 15) quails developed any clinical signs. In contrast to the low virus shedding titers observed from the swH1N1-inoculated quails, birds inoculated with dkH3N2 and pH1N1 shed relatively high titers of virus predominantly from the respiratory tract until 5 and 7 DPI, respectively, that were rarely transmitted to the contact quails. Gross and histopathological lesions were observed in the respiratory and intestinal tracts of quail inoculated with either pH1N1 or dkH3N2, indicating that these viruses were more pathogenic than swH1N1. Sero-conversions were detected 7 DPI in two out of five pH1N1-inoculated quails, three out of five quails inoculated with swH1N1, and four out of five swH1N1-infected contact birds. Taken together, this study demonstrated that quails were more susceptible to infection with pH1N1 and dkH3N2 than swH1N1.

Quail as a potential mixing vessel for the generation of new reassortant influenza A viruses.

Quail has been proposed as one of the intermediate hosts supporting the generation of newly reassortant influenza A viruses (IAVs) with the potential to infect humans. To evaluate the role of quail as an intermediate host of IAVs, co-infections of quail with swine-origin pandemic H1N1 2009 (pH1N1) and low pathogenic avian influenza (LPAI) duck H3N2 (dkH3N2) viruses (n=10) or endemic Thai swine H1N1 (swH1N1) and dkH3N2 viruses (n=10) were conducted. Three additional groups of five quail were each inoculated with pH1N1, swH1N1 and dkH3N2 as control groups to verify that each virus can infect quail. Our result showed that co-infected quail shed higher viral titers from the respiratory tract than single virus infected quail. This study confirmed that reassortant viruses could be readily generated in the respiratory tract of quail from both the pH1N1/dkH3N2 co-infected group (100% of quail generating reassortant viruses) and the swH1N1/dkH3N2 (33% of quail generating reassortant viruses) co-infected group without discernible clinical signs. The reassortment efficacy between the two combination of viruses was different in that the frequency of reassortant viruses was significantly higher in pH1N1/dkH3N2 co-infected quail (21.4%) compared to swH1N1/dkH3N2 co-infected quail (0.8%), indicating that gene combinations in pH1N1 have a higher potential to reassort with dkH3N2 compared to swH1N1. In summary, our result confirmed that quail could be an intermediate host of IAVs for generating new reassortant viruses. Our finding highlights the importance of monitoring IAVs especially pH1N1 in quail.