First molecular detection of porcine circovirus type 4 (PCV4) in Malaysia.

Porcine circovirus type 4 (PCV4) is the newest member in the porcine circovirus family, first reported in 2020. To date, the presence of PCV4 has only been reported in China, South Korea and most recently in Thailand. Detection of PCV4 have been reported in various production stages of pigs from piglets, finishers to sows; associated with a myriad of clinical manifestations including porcine dermatitis and nephropathy syndrome (PDNS), postweaning multisystemic wasting syndrome (PMWS), respiratory, enteric and neurological diseases. While successful virus isolation and culture has yet to be reported, pathogenicity of PCV4 has been demonstrated through infectious clone studies. The objective of this study is to investigate the presence of PCV4 in Malaysian porcine population to update the epidemiology of porcine circoviruses in Malaysia. A total of 49 samples from commercial intensive pig farms, abattoir and wild boar population were subjected to conventional polymerase chain reaction assay to detect PCV4 capsid (cap) genome. Resulting cap nucleotide sequences were analyzed for maximum likelihood phylogeny relationship. Results revealed that PCV4 is present in Peninsular Malaysia at a molecular prevalence of 4.08% (2 / 49 samples). Both PCV4 positive samples originated from clinically healthy finishers. Malaysian PCV4 strains were classified as genotype PCV4b, and were found to be phylogenetically distinct from the China, South Korea and Thailand strains. With this latest update of the novel PCV4 in Malaysia, it is clear that more attention needs to be given to the investigation of novel porcine circoviruses (PCV) and management of PCV diseases.

The effect of feeding black soldier fly larvae on growth performance, protein, and fat content of red hybrid tilapia (Oreochromis spp.).

Background and Aim: In the aquaculture industry, the crucial goal is to minimize production costs, especially feeding costs, without significant side effects. Black soldier fly larva (BSFL) is a locally available, eco-friendly, and sustainable source that is high in crude protein (42% dry matter [DM]) and fat (35% DM). This study aimed to determine the growth performance along with the composition of crude fat and protein in red hybrid fingerlings after the addition of BSFL into the diet. Materials and Methods: A total of 120 fingerlings of uniform size (mean initial weight of 1.46 ± 0.06 g) were randomly assigned to one of four groups (n = 10) (A, B, C, and D) per tank (1 m × 2 m × 1 m). For 21 days, Group A (control group) was fed with 100% commercial diet; Group B was fed with 90% commercial fish diet + 10% BSFL; Group C was fed with 80% commercial fish diet + 20% BSFL; and Group D was fed with 70% commercial fish diet + 30% BSFL. Feed efficiency, growth performance, and proximate composition analysis were performed on the fish. Results: The results displayed that the group with the highest BSFL percentage had a greater effect on protein and fat composition than the control group. The proximate composition analysis of fish-fed diet revealed that an increase in the level of BSFL inclusion increases the protein content in the fish. In comparison to the other groups, the experimental diet with 30% BSFL inclusion has the highest levels of crude protein (80.30% DM) and fat (2.90% DM). Conclusion: It is concluded that incorporating BSFL into a commercial diet for red hybrid tilapia fingerlings increased crude protein and fat composition, providing an alternative protein and fat source in fish diets.

Evidence for Nipah virus recrudescence and serological patterns of captive Pteropus vampyrus.

This study aimed to describe the transmission dynamics, the serological and virus excretion patterns of Nipah virus (NiV) in Pteropus vampyrus bats. Bats in captivity were sampled every 7-21 days over a 1-year period. The data revealed five NiV serological patterns categorized as high and low positives, waning, decreasing and increasing, and negative in these individuals. The findings strongly suggest that NiV circulates in wild bat populations and that antibody could be maintained for long periods. The study also found that pup and juvenile bats from seropositive dams tested seropositive, indicating that maternal antibodies against NiV are transmitted passively, and in this study population may last up to 14 months. NiV was isolated from the urine of one bat, and within a few weeks, two other seronegative bats seroconverted. Based on the temporal cluster of seroconversion, we strongly believe that the NiV isolated was recrudesced and then transmitted horizontally between bats during the study period.

Sequence and phylogenetic analysis of S1, S2, M, and N genes of infectious bronchitis virus isolates from Malaysia.

Two Malaysian infectious bronchitis virus isolates, MH5365/95 and V9/04 were characterized based on sequence and phylogenetic analyses of S1, S2, M, and N genes. Nucleotide sequence alignments revealed many point mutations, short deletions, and insertions in S1 region of both IBV isolates. Phylogenetic analysis of S1 gene and sequences analysis of M gene indicated that MH5365/95 and V9/04 belong to non-Massachusetts strain. However, both isolates share only 77% identity. Analysis based on S1 gene showed that MH5365/95 shared more than 87% identity to several Chinese strains. Meanwhile, V9/04 showed only 67-77% identity to all the previously studied IBV strains included in this study suggesting it is a variant of IBV isolate that is unique to Malaysia. Phylogenetic analysis suggests, although both isolates were isolated 10 years apart from different states in Malaysia, they shared a common origin. Analysis based on S2 and N genes indicated that both strains are highly related to each other, and there are fewer mutations which occurred in the respective genes.

Detection of avian leukosis virus subgroup J in chicken flocks from Malaysia and their molecular characterization.

Previously we have shown that avian leukosis virus subgroup J (ALV-J) might be present in chicken flocks from Malaysia based on serological study and also on detection of tissue samples with myelocytic infiltration. In this study, the polymerase chain reaction was used to detect ALV-J sequences from archived frozen samples. Out of 21 tissue samples examined, 16 samples were positive for proviral DNA and four samples for ALV-J RNA. However, only nine samples were found positive for myelocytic infiltration. A total of 465 base pairs equivalent to positions 5305 to 5769 of HPRS-103 from each of the viral RNA positive samples were characterized. Sequence analysis indicated that the samples showed high identity (95.9 to 98.2%) and were close to HPRS-103 with identities between 97.4 and 99.3%. This study indicates that ALV-J-specific sequences can be detected by polymerase chain reaction from frozen tissue samples with and without myelocytic infiltration.

Performance of an RT-nested PCR ELISA for detection of Newcastle disease virus.

A sensitive and specific RT-nested PCR coupled with an ELISA detection system for detecting Newcastle disease virus is described. Two nested pairs of primer which were highly specific to all the three different pathotypes of NDV were designed from the consensus fusion gene sequence. No cross-reactions with other avian infectious agents such as infectious bronchitis virus, infectious bursal disease virus, influenza virus, and fowl pox virus were observed. Based on agarose electrophoresis detection, the RT-nested PCR was about 100 times more sensitive compared to that of a non-nested RT-PCR. To facilitate the detection of the PCR product, an ELISA detection method was then developed to detect the amplified PCR products and it was shown to be ten times more sensitive than gel electrophoresis. The efficacy of the nested PCR-ELISA was also compared with the conventional NDV detection method (HA test) and non-nested RT-PCR by testing against a total of 35 tissue specimens collected from ND-symptomatic chickens. The RT-nested PCR ELISA found NDV positive in 21 (60%) tissue specimens, while only eight (22.9%) and two (5.7%) out of 35 tissue specimens were tested NDV positive by both the non-nested RT-PCR and conventional HA test, respectively. Due to its high sensitivity for the detection of NDV from tissue specimens, this PCR-ELISA based diagnostic test may be useful for screening large number of samples.

Tropism of subgroup J avian leukosis virus as detected by in situ hybridization.

The HPRS-103 strain of avian retrovirus is the prototype of subgroup J avian leukosis virus (ALV-J) and causes myeloid leukosis in meat-type chickens. Using immunohistochemical detection of the viral groupspecific antigen (Gag) we have previously demonstrated that the induction of myeloid leukosis by ALV-J is associated with viral tropism for myelomonocytic cells. In this paper we describe an in situ hybridization (ISH) technique using digoxigenin (DIG)-labelled probes for detecting RNA transcripts in tissues from chickens infected with avian leukosis viruses (ALV) of subgroups J (HPRS-103 strain) and A (RAV-1 strain). Virus-specific RNA was detected mainly in the heart, kidney, proventriculus and adrenal in locations similar to those of the Gag protein. Viral gene expression could not be detected in the bone marrow or tumour tissues using this test. Higher levels of viral gene expression in the bursa of Fabricius infected with RAV-1, but not with HPRS-103, might help explain the inability of the latter virus to induce lymphoid leukosis.

Development and application of polymerase chain reaction (PCR) tests for the detection of subgroup J avian leukosis virus.

Subgroup J avian leukosis virus (ALV) is a recently identified avian retrovirus associated with myeloid leukosis in meat-type chickens. The env gene of the HPRS-103 strain of ALV, the prototype of this subgroup, differs considerably from that of other subgroups, but shows close homology to the env-like sequences of members of the EAV family of endogenous retroviruses. Polymerase chain reaction (PCR) tests using two sets of primers were developed for the specific detection of the members of this new subgroup along with another pair of primers for detecting other subgroup viruses. The specificity and sensitivity of this detection system was compared with the conventional detection methods in experimentally and naturally infected samples. The use of PCR was found to be rapid, specific and more sensitive than the conventional diagnostic tests for the detection of ALV. Moreover, the two subgroup J ALV-specific PCR tests were found to be capable of differentiating between 'prototype-like' viruses and more recent isolates which show extensive antigenic and sequence variations. The use of this test as a rapid and sensitive method of detection of viruses in epidemiological studies and eradication programs is discussed.

Tissue tropism of the HPRS-103 strain of J subgroup avian leukosis virus and of a derivative acutely transforming virus.

The tissue tropism was studied for the HPRS-103 strain of avian leukosis virus, which belongs to a new envelope subgroup, designated J. Studies were conducted in blood monocyte and bone marrow cell cultures and in chickens from six lines that had been shown previously to differ in susceptibility to induction by this virus of myeloid leukosis and other tumors. Using an immunohistochemical technique to detect expression of viral group-specific antigen (Gag) in various tissues, we detected no major differences among the six lines of chickens at 3 and 7 weeks of age following infection as embryos. Thus, Gag expression did not correlate with differences in tumor susceptibility. Of the tissues examined, greatest Gag expression was observed in cells specific to the adrenal gland, heart, kidney, proventriculus and especially in smooth muscle cells and connective tissue. After infection of 1-day-old chicks, greater tissue expression was observed in line 21 chicks, which mostly developed a tolerant viremic infection, than in Brown Leghorn chicks, which developed virus-neutralizing antibodies. An acutely transforming virus, strain 966, derived from HPRS-103-induced myeloid leukosis, showed a tropism similar to HPRS-103. The HPRS-103 strain showed a lower propensity to replicate in the medullary region of the lymphoid follicles of the bursa of Fabricius than did the RAV-1 strain of subgroup A avian leukosis virus. This low bursal tropism may be a factor in why HPRS-103 does not induce lymphoid leukosis. The HPRS-103 and 966 virus replicated in blood monocyte cultures from chickens from the six lines, indicating a tropism for the myelomonocytic cell lineage. In comparison, as previously reported, RAV-1 did not replicate well in the monocyte cultures, whereas RAV-2, a subgroup B avian leukosis virus, did replicate. The tropism of HPRS-103 for monocytes may relate to its ability to cause myeloid leukosis. Monocyte and bone marrow cell cultures from the six lines ranked similarly in differences in susceptibility to transformation by 966 virus and showed evidence that their relative susceptibilities correlated with susceptibility of chickens from these lines to induction of myeloid leukosis by HPRS-103, suggesting common tissue-specific viral and host factors involved in oncogenesis by these two viruses.

A low incidence of histiocytic sarcomatosis associated with infection of chickens with the HPRS-103 strain of subgroup J avian leukosis virus.

Ten cases of histiocytic proliferative lesions in meat-type chickens associated in low incidence with infection by subgroup J avian leukosis virus (ALV) are described. Six were field cases in adult chickens from naturally infected flocks and four were from younger birds from transmission experiments with HPRS-103 ALV or the related acutely transforming ALV strains 17 and 879. The lesions were observed mostly in the spleen and in some cases in other organs. Microscopically, the lesions were comprised mainly of pleomorphic histiocyte-like cells admixed with variable numbers of lymphoid cells. More detailed studies were carried out on two birds at 4 and 7 wk of age following infection with HPRS-103 at 1 day of age. These birds had multiple small nodular lesions in the spleen, liver, and kidney that appeared similar cytologically to the more extensive lesions in older birds. Monoclonal antibodies specific for various lymphoid and nonlymphoid accessory cells were used in immunohistochemical studies to identify a predominance of cells of monocyte/macrophage lineage, and CD4- and CD8-positive lymphocytes, in the splenic nodules. Ultrastructural studies also revealed a similar mixed population of cells. Expression of ALV group-specific antigen, and gag and ALV-J env RNA, was not a marked feature of the histiocytic lesions. The proliferative histiocytic lesion is designated a histiocytic sarcomatosis.