Molecular investigation of feline coronavirus (FCoV) in local pet cats

Feline coronavirus (FCoV) infection is a very common in cat population. FCoV is further classified into two biotypes namely feline enteric coronavirus (FECV) and mutated feline infectious peritonitis virus (FIPV), in which FIPV causes a fatal immune complex disease by changing the tropism from enterocytes to monocytes. Previous studies on molecular detection of FCoV in cats were carried out in catteries but limited study investigate the presence of FCoV antigen in local pet cats. By considering this fact, this study aims to detect FCoV antigen via RT-PCR assay in local pet cats and to compare the similarity of the identified FCoV strain with previous related virus by phylogenetic analysis. By using convenience sampling, rectal swabs and buffy coat were collected from 16 clinically ill pet cats and 5 healthy pet cats. Viral RNA was extracted and subjected to one-step RT-PCR, targeting polymerase gene. Only one out of 21 fecal samples was positive for FCoV and none from buffy coat samples. Phylogenetic analysis revealed that the identified positive sample was highly homologous, up to 95%, to FCoV strain from Netherlands and South Korea on partial sequence of polymerase gene. In conclusion, this study detected FCoV antigen in local pet cats from fecal samples while negative detection from fecal and buffy coat samples could not completely rule out the possibilities of FCoV infection due to the complexity of the virus diagnosis that require multiple series of analysis

West Nile virus infection in human and animals: potential risks in Malaysia

West Nile virus (WNV) is a zoonotic RNA virus maintained in enzootic cycles involving mosquito mainly Culex and Aedes spp. as the vector and birds as the reservoir host. WNV is endemic in Africa, Europe and Western Asia. Human infection results in asymptomatic illness such as fever, headache, tiredness, body aches, nausea, vomiting, skin rash, swollen lymph glands and neuroinvasive disease are seen in less than 1% of infected persons. The spectrum of symptoms in animals includes fever, weakness and paralysis of hind limbs, impaired vision, ataxia, head pressing, aimless wandering, seizures, inability to swallow, walking in circles and coma. Based on the previous study in Malaysia, the antibody and nucleic acid against WNV were detected in Orang Asli, captive birds and horses. In this paper, potential risk factors contributing to WNV occurrence are discussed. How the disease or infection is diagnosed and controlled was also discussed

Bursal immunopathology responses of specific-pathogen-free chickens and red jungle fowl infected with very virulent infectious bursal disease virus

Very virulent infectious bursal disease virus (vvIBDV) targets B lymphocytes in the bursa of Fabricius (BF), causing immunosuppression and increased mortality rates in young birds. There have been few studies on the host immune response following vvIBDV infection at different inoculum doses in chickens with different genetic backgrounds. In this study, we characterized the immune responses of specific-pathogen-free (SPF) chickens and Malaysian red jungle fowl following infection with vvIBDV strain UPM0081 at 103.8 and 106.8 times the 50% embryo infectious dose (EID50). The viral burden, histopathological changes, immune cell populations, and expression of immune-related genes were measured and compared between infected and uninfected bursa at specific intervals. The populations of KUL1+, CD3+CD4+ and CD3+CD8+ cells were significantly increased in both types of chickens at 3 dpi, and there was significant early depletion of IgM+ B cells at 1 dpi in the red jungle fowl. vvIBDV infection also induced differential expression of genes that are involved in Th1 and pro-inflammatory responses, with groups receiving the higher dose (106.8 EID50) showing earlier expression of IFNG, IL12B, IL15, IL6, CXCLi2, IL28B, and TLR3 at 1 dpi. Although both chicken types showed equal susceptibility to infection, the red jungle fowl were clinically healthier than the SPF chickens despite showing more depletion of IgM+ B cells and failure to induce IFNB activation. In conclusion, high-dose vvIBDV infection caused an intense early host immune response in the infected bursa, with depletion of IgM+ B cells, bursal lesions, and cytokine expression as a response to mitigate the severity of the infection

Serological survey of Aujeszkys disease in Peninsular Malaysia in 2016

Aujeszky’s disease (AD) is a common swine disease that widespread throughout the world. The symptoms include nervous signs, respiration and reproduction problems that lead to great economic losses to the industry. AD is endemic in Malaysia, where outbreaks had been reported in previous years. In Malaysia, approximately 95% of the pig farms are vaccinated for AD. Despite the regular vaccination, AD serological status remains unknown in this country. This study provides AD serological status in Peninsular Malaysia in 2016 based on the samples submitted to Faculty of Veterinary Medicine, University Putra Malaysia (UPM). A total of 1154 serum samples from 36 farms were submitted for AD ELISA diagnostic test; grouped as 8 weeks, 12 weeks, 16 weeks, 20 weeks, gilts and sows with different parity. The samples were subjected to AD antibody detection with IDEXX Pseudorabies Virus gpI Antibody Test Kit. Among the 36 farms submitted to UPM, 8 farms were detected with positive gI antibody indicated that these farms were still facing challenges from AD field virus. Among these eight seropositive farms, three farms were having seroprevalence in the range of 33.33% to 37.14%. In general, vaccination of AD is ideal and stable in Malaysia but we still need to be alert with the field challenge as it will be a threat to the industry

Evidence of West Nile virus infection in migratory and resident wild birds in west coast of peninsular Malaysia

West Nile virus (WNV) is a zoonotic mosquito-borne flavivirus that is harbored and amplified by wild birds via the enzootic transmission cycle. Wide range of hosts are found to be susceptible to WNV infection including mammals, amphibians and reptiles across the world. Several studies have demonstrated that WNV was present in the Malaysian Orang Asli and captive birds. However, no data are available on the WNV prevalence in wild birds found in Malaysia. Therefore this study was conducted to determine the serological and molecular prevalence of WNV in wild birds in selected areas in the West Coast of Peninsular Malaysia. Two types of wild birds were screened, namely migratory and resident birds in order to explore any possibility of WNV transmission from the migratory birds to the resident birds. Thus, a cross-sectional study was conducted at the migratory birds sanctuary located in Kuala Gula, Perak and Kapar, Selangor by catching 163 migratory birds, and 97 resident birds from Kuala Gula and Parit Buntar, Perak at different time between 2016 and 2017 (Total, n = 260). Blood and oropharyngeal swabs were collected for serological and molecular analysis, respectively. Serum were screened for WNV antibodies using a commercial competitive ELISA (c-ELISA) (ID Screen® West Nile Competition Multi-species ELISA, ID VET, Montpellier, France) and cross-reactivity towards Japanese Encephalitis virus (JEV) was also carried out using the JEV-double antigen sandwich (DAS) ELISA. Oropharyngeal swabs were subjected to one-step RT-PCR to detect WNV RNA, in which positive reactions were subsequently sequenced. WNV seropositive rate of 18.71% (29/155) at 95% CI (0.131 to 0.260) and molecular prevalence of 15.2% (16/105) at 95% CI (0.092 to 0.239) were demonstrated in migratory and resident wild birds found in West Coast Malaysia. Phylogenetic analyses of the 16 WNV isolates found in this study revealed that the local strains have 99% similarity to the strains from South Africa and were clustered under lineage 2. Evidence of WNV infection in resident and migratory birds were demonstrated in this study. As a summary, intervention between migratory birds, resident birds and mosquitoes might cause the introduction and maintenance of WNV in Malaysia, however the assumption could be further proven by studying the infection dynamics in the mosquitoes present in the studied areas

Exposure to Zoonotic West Nile Virus in Long-Tailed Macaques and Bats in Peninsular Malaysia

The role of wildlife such as wild birds, macaques, and bats in the spreading and maintenance of deadly zoonotic pathogens in nature have been well documented in many parts of the world. One such pathogen is the mosquitoes borne virus, namely the West Nile Virus (WNV). Previous research has shown that 1:7 and 1:6 Malaysian wild birds are WNV antibody and RNA positive, respectively, and bats in North America may not be susceptible to the WNV infection. This study was conducted to determine the status of WNV in Malaysian macaques and bats found in mangrove forests and caves, respectively. Archive sera and oropharyngeal swabs from long-tailed macaques were subjected to the antibody detection using WNV competitive enzyme-linked immunosorbent assay (c-ELISA) and WNV RNA using RT-PCR, respectively, while the archive oropharyngeal and rectal swabs from bats were subjected to RT-PCR without serological analysis due to the unavailability of serum samples. The analysis revealed a WNV seropositivity of 29.63% (24/81) and none of the macaques were positive for WNV RNA. Meanwhile, 12.2% (5/41) of the bats from Pteropodidae, Emballonuridae, and Rhinolophidae families tested positive for WNV RNA. Here, we show a high WNV antibody prevalence in macaques and a moderate WNV RNA in various Malaysian bat species, suggesting that WNV circulates through Malaysian wild animals and Malaysian bat species may be susceptible to the WNV infectio

Differential modulation of immune response and cytokine profiles in the bursae and spleen of chickens infected with very virulent infectious bursal disease virus

Background: Very virulent infectious bursal disease virus (vvIBDV) induces immunosuppression and inflammation in young birds, which subsequently leads to high mortality. In addition, infectious bursal disease (IBD) is one of the leading causes of vaccine failure on farms. Therefore, understanding the immunopathogenesis of IBDV in both the spleen and the bursae could help effective vaccine development. However, previous studies only profiled the differential expression of a limited number of cytokines, in either the spleen or the bursae of Fabricius of IBDV-infected chickens. Thus, this study aims to evaluate the in vitro and in vivo immunoregulatory effects of vvIBDV infection on macrophage-like cells, spleen and bursae of Fabricius. Results: The viral load was increased during the progression of the in vitro infection in the HD11 macrophage cell line and in vivo, but no significant difference was observed between the spleen and the bursae tissue. vvIBDV infection induced the expression of pro-inflammatory and Th1 cytokines, and chemokines from HD11 cells in a time- and dosage-dependent manner. Furthermore, alterations in the lymphocyte populations, cytokine and chemokine expression, were observed in the vvIBDV-infected spleens and bursae. A drastic rise was detected in numbers of macrophages and pro-inflammatory cytokine expression in the spleen, as early as 2 days post-infection (dpi). On 4 dpi, macrophage and T lymphocyte infiltration, associated with the peak expression of pro-inflammatory cytokines in the bursae tissues of infected chickens were observed. The majority of the significantly regulated pro-inflammatory cytokines and chemokines, in vvIBDV-infected spleens and bursae, were also detected in vvIBDV-infected HD11 cells. This cellular infiltration subsequently resulted in a sharp rise in nitric oxide (NO) and lipid peroxidation levels. Conclusion: This study suggests that macrophage may play an important role in regulating the early expression of pro-inflammatory cytokines, first in the spleen and then in the bursae, the latter tissue undergoing macrophage infiltration at 4 dpi

Comparison of expression for IL-15 and IL-18 on dendritic cells and macrophages upon infection with recombinant fowlpox virus versus wild type fowlpox virus

Fowlpox is a viral disease of chicken caused by fowlpox virus (FWPV). Compared to other viral infectious diseases, studies on the interaction of antigen-presenting cells (APC) such as dendritic cells (DCs) and macrophage with FWPV remains limited. Therefore, this work was aimed to characterize cytokine responses upon infection of recombinant fowlpox viruses (rFWPV) expressing H5 gene of avian influenza virus (AIV) compared to wild type FWPV on chicken DCs and macrophage cells. Chicken bone marrow cells were first isolated. The cells were differentiated into DCs (GM-CSF and IL-4 acted as supplements in RPMI media), or into macrophages (no supplements added). In order to quantify chicken IL-15 and IL-18 cytokine expression, qPCR assays based on real-time analysis were performed. Data suggested that rFWPV induced higher expression for IL-15 and IL-18 on macrophage while wild type FWPV induced higher expression for IL-15 and IL-18 on DC

Isolation and genetic characterization of canine parvovirus CPV in a Malayan tiger

Naïve Felidae in the wild may harbor infectious viruses of importance due to cross-species transmission between the domesticated animals or human–wildlife contact. However, limited information is available on virus shedding or viremia in the captive wild felids, especially in Malaysia. Four infectious viruses of cat, feline herpesvirus (FHV), feline calicivirus (FCV), canine distemper virus (CDV), and canine parvovirus (CPV), were screened in leopards, feral cats, and tigers in Malaysia based on virus isolation in Crandell-Rees feline kidney (CRFK) cells, PCR/RT-PCR, and whole-genome sequencing analysis of the positive isolate. From a total of 36 sera collected, 11 samples showed three consecutive cytopathic effects in the cell culture and were subjected to PCR using specific primers for FHV, FCV, CDV, and CPV. Only one sample from a Malayan tiger was detected positive for CPV. The entire viral genome of CPV (UPM-CPV15/P. tigris jacksoni; GenBank Accession number MW380384) was amplified using the Sanger sequencing approach. Genome sequencing of the isolate revealed 99.13, 98.65, and 98.40% close similarity to CPV-31, CPV-d Cornell #320, and CPV-15 strains, respectively, and classified as CPV-2a. Time-scaled Bayesian Maximum Clade Credibility tree for the non-structural (NS) genes of CPV showed a close relationship to the isolates CPV-CN SD6_2014 and KSU7-SD_2004 from China and USA, respectively, while the capsid gene showed the same ancestor as the FPV-BJ04 strain from China. The higher evolution rate of the capsid protein (CP) (VP 1 and VP2) [1.649 × 10−5 (95% HPD: 7.626 × 10−3 to 7.440 × 10−3)] as compared to the NS gene [1.203 × 10−4 (95% HPD: 6.663 × 10−3 to 6.593 × 10−3)] was observed in the CPV from this study, and fairly higher than other parvovirus species from the Protoparvovirus genus. Genome sequencing of the isolated CPV from a Malayan tiger in the present study provides valuable information about the genomic characteristics of captive wild felids, which may add information on the presence of CPV in species other than dogs

Canine parvovirus (CPV) infection in a great dane puppy: a case report

Canine parvovirus (CPV) infection is a major disease affecting young pups with high contagiousness and mortality worldwide. Unvaccinated pups between 6 weeks to 6 months are most susceptible to CPV infection. It manifests into enteritis and myocarditis forms. Classical signs of enteritis form include acute onset of vomiting, haemorrhagic diarrhoea, and pyrexia. A case of unvaccinated 3-month-old female Great Dane puppy was presented with complaints of hematemesis, hematochezia and shooting diarrhoea. A tentative diagnosis of CPV was made. It was humanely euthanised due to chronic body weight loss and poor response to treatments. Post-mortem examinations revealed congested lung with frothy exudate, whitish plaques on heart coronary groove, thickened intestinal mucosa, and generalised reddening in the liver and kidneys. Microscopic changes revealed interstitial pneumonia with edema, lymphocytic myocarditis, viral enteritis with villous atrophy, and liver congestion. A faecal samples for polymerase chain reaction (PCR) method revealed positive result for parvovirus. Based on pathological and PCR findings, it was definitively diagnosed with CPV infection. The chance of its survival without aggressive treatment remains low. Treatment and management are still limited to supportive care without existing agent-specific treatment. Therefore, CPV infection should be controlled and prevented by providing vaccination to the pups from the age of 6 weeks