The descriptive distribution and phylogeny of feline coronaviruses (FCoVs) were studied in cats suspected of having feline infectious peritonitis (FIP) in Malaysia. Ascitic fluids and/or biopsy samples were subjected to a reverse transcription polymerase chain reaction (RT-PCR) targeted for a conserved region of 3'untranslated region (3'UTR) of the FCoV genome. Eighty nine percent of the sampled animals were positive for the presence of FCoV. Among the FCoV positive cats, 80% of cats were males and 64% were below 2 years of age. The FCoV positive cases included 56% domestic short hair (DSH), 40% Persian, and 4% Siamese cats. The nucleotide sequences of 10 selected amplified products from FIP cases were determined. The sequence comparison revealed that the field isolates had 96% homology with a few point mutations. The extent of homology decreased to 93% when compared with reference strains. The overall branching pattern of phylogenetic tree showed two distinct clusters, where all Malaysian isolates fall into one main genetic cluster. These findings provided the first genetic information of FCoV in Malaysia.
Infectious bursal disease is a highly contagious disease in the poultry industry and causes immunosuppression in chickens. Genome-wide regulations of immune response genes of inbred chickens with different genetic backgrounds, following very virulent infectious bursal disease virus (vvIBDV) infection are poorly characterized. Therefore, this study aims to analyse the bursal tissue transcriptome of six inbred chicken lines 6, 7, 15, N, O and P following infection with vvIBDV strain UK661 using strand-specific next-generation sequencing, by highlighting important genes and pathways involved in the infected chicken during peak infection at 3 days post-infection. All infected chickens succumbed to the infection without major variations among the different lines. However, based on the viral loads and bursal lesion scoring, lines P and 6 can be considered as the most susceptible lines, while lines 15 and N were regarded as the least affected lines. Transcriptome profiling of the bursa identified 4588 genes to be differentially expressed, with 2985 upregulated and 1642 downregulated genes, in which these genes were commonly or uniquely detected in all or several infected lines. Genes that were upregulated are primarily pro-inflammatory cytokines, chemokines and IFN-related. Various genes that are associated with B-cell functions and genes related to apoptosis were downregulated, together with the genes involved in p53 signalling. In conclusion, bursal transcriptome profiles of different inbred lines showed differential expressions of pro-inflammatory cytokines and chemokines, Th1 cytokines, JAK-STAT signalling genes, MAPK signalling genes, and their related pathways following vvIBDV infection.
BACKGROUND:
Feline Infectious Peritonitis (FIP) is a lethal systemic disease, caused by the FIP Virus (FIPV); a virulent mutant of Feline Enteric Coronavirus (FECV). Currently, the viruses virulence determinants and host gene expressions during FIPV infection are not fully understood.
METHODS:
RNA sequencing of Crandell Rees Feline Kidney (CRFK) cells, infected with FIPV strain 79-1146 at 3 hours post infection (h.p.i), were sequenced using the Illumina next generation sequencing approach. Bioinformatic's analysis, based on Felis catus 2X annotated shotgun reference genome, using CLC bio Genome Workbench mapped both control and infected cell reads to 18899 genes out of 19046 annotated genes. Kal's Z test statistical analysis was used to analyse the differentially expressed genes from the infected CRFK cells. Real time RT-qPCR was developed for further transcriptional profiling of three genes (PD-1, PD-L1 and A3H) in infected CRFK cells and Peripheral Blood Mononuclear Cells (PBMCs) from healthy and FIP-diseased cats.
RESULTS:
Based on Kal's Z-test, with False Discovery Rate (FDR) <0.05 and >1.99 fold change on gene expressions, a total of 61 genes were differentially expressed by both samples, where 44 genes were up-regulated and the remainder were down-regulated. Most genes were closely clustered together, suggesting a homogeneous expression. The majority of the genes that were significantly regulated, were those associated with monocytes-macrophage and Th1 cell functions, and the regulation of apoptosis. Real time RT-qPCR developed focusing on 2 up-regulated genes (PD-L1 and A3H) together with an apoptosis associated gene PD-1 expressions in FIPV infected CRFK cells and in PBMCs from healthy and FIP diagnosed cats produced concordant results with transcriptome data.
CONCLUSION:
The possible roles of these genes, and their importance in feline coronaviruses infection, are discussed.
Newcastle disease (ND) is one of the most important avian diseases with considerable threat to the productivity of poultry all over the world. The disease is associated with severe respiratory, gastrointestinal, and neurological lesions in chicken leading to high mortality and several other production related losses. The aetiology of the disease is an avian paramyxovirus type-1 or Newcastle disease virus (NDV), whose isolates are serologically grouped into a single serotype but genetically classified into a total of 19 genotypes, owing to the continuous emergence and evolution of the virus. In Nigeria, molecular characterization of NDV is generally very scanty and majorly focuses on the amplification of the partial F gene for genotype assignment. However, with the introduction of the most objective NDV genotyping criteria which utilize complete fusion protein coding sequences in phylogenetic taxonomy, the enormous genetic diversity of the virus in Nigeria became very conspicuous. In this review, we examine the current ecological distribution of various NDV genotypes in Nigeria based on the available complete fusion protein nucleotide sequences (1662 bp) in the NCBI database. We then discuss the challenges of ND control as a result of the wide genetic distance between the currently circulating NDV isolates and the commonest vaccines used to combat the disease in the country. Finally, we suggest future directions in the war against the economically devastating ND in Nigeria.
Infectious bursal disease is one of an OIE list of notifiable diseases. Chicken is the only host that manifests clinical signs and its pathogenicity is correlated with the distribution of antigens in organs. This study was conducted to determine disease pathogenesis and virus tissue tropism by in situ PCR, immunoperoxidase staining (IPS), and HE staining. Twenty four chickens were infected with very virulent Infectious Bursal Disease Virus (vvIBDV). Fifteen chickens were kept as a control group. Infected chickens were sacrificed at hrs 2, 4, 6, 12, days 1, 2, 4, and 6 post-inoculation (pi). While, control chickens were euthanized on days 0, 1, 2, 4, and 6 pi. Different tissues were collected, fixed in 10% buffered formalin, and processed. At hr 2 pi, virus was detected in intestinal, junction of the proventriculus and gizzard, cecal tonsil, liver, kidney, and bursa of Fabricius. At hr 4 pi, virus reached spleen, and at hr 6 pi, it entered thymus. At hr 12 pi, virus concentration increased in positive tissues. The latest invaded tissue was muscle on day 1 pi. Secondary viraemia occurred during 12-24 h pi. In situ PCR was the most sensitive technique to highlight obscure points of infection in this study.
Infectious bronchitis viruses (IBVs) circulating in Malaysia are classified into two groups as Malaysian QX-like and variant strains. In this study, the pathogenicity of IBS130/2015 (QX-like) and IBS037A/2014 (variant) IBVs in 1-day-old and 30-day-old specific pathogen free (SPF) chickens was characterized. Both strains caused respiratory and kidney infections based on immunohistochemistry (IHC), real-time quantitative polymerase chain reaction (qPCR) and a ciliostasis study; however, the results showed that the QX-like strain was more pathogenic, caused higher mortality and showed higher tissue tropism for the kidney than the variant strain. In contrast, despite causing low or no mortality depending on the age of the infected chickens, the Malaysian variant strain showed high tissue tropism for the respiratory tract compared with the QX-like strain. IHC and qPCR indicated the presence of both IBV strains in the epithelial lining of villi in the jejunum and the caecal tonsil; however, no pathological changes were detected in these organs. Both the Malaysian QX-like and variant IBV strains are able to infect the respiratory tract and kidney of chickens irrespective of age.
Limited deep studies are available in the field of early stages of pathogenesis of Newcastle disease virus (NDV) infection and tissue tropism of NDV. In this study, 24 specific pathogen free (SPF) chickens of white leghorn breed were infected with Newcastle disease (ND) by intranasal administration of 10⁵ 50% EID50/0.1 mL of velogenic NDV (vNDV). A second group of 15 chickens were kept as a control group. Chickens were monitored every day to record clinical signs. Infected chickens were euthanized by cervical dislocation at successive times, namely at hours (hrs) 2, 4, 6, 12, days 1, 2, 4, and 6 post-inoculation (pi). Whereas, control group chickens were euthanized on days 0, 1, 2, 4, and 6 pi. Tissues of brain, trachea, lung, caecal tonsil, liver, kidney, spleen, heart, proventriculus, intestine, and thymus were collected, fixed in 10% buffered formalin, embedded in paraffin, and sectioned. HS staining, immunoperoxidase staining (IPS) and in situ PCR were applied. It was concluded that at hr 2 pi, virus seemed to be inclined to trachea and respiratory tract. Meanwhile, it attacked caecal tonsils, intestine and bursa of Fabricus. While primary viraemia was ongoing, virus created footing in kidney and thymus. At hr 4 pi, proventriculus, liver, and spleen were attacked. However, at hr 6 pi, brain and heart were involved. Secondary viraemia probably started as early as hr 12 pi since all collected tissues were positive. Tissue tropism was determined in trachea, caecal tonsil, liver, bursa of Fabricius, intestine, proventriculus, lung, spleen, thymus, kidney, heart, and brain.
In this study, a Salmonella Typhimurium lytic bacteriophage, Φ st1, which was isolated from chicken faecal material, was evaluated as a candidate for biocontrol of Salmonella in chickens. The morphology of Φ st1 showed strong resemblance to members of the Siphoviridae family. Φ st1 was observed to be a DNA phage with an estimated genome size of 121 kbp. It was found to be able to infect S. Typhimurium and S. Hadar, with a stronger lytic activity against the former. Subsequent characterisation of Φ st1 against S. Typhimurium showed that Φ st1 has a latent period of 40 min with an average burst size of 22 particles per infective centre. Approximately 86.1% of the phage adsorbed to the host cells within the initial 5 min of infection. At the optimum multiplicity of infection (MOI) (0.1), the highest reduction rate of S. Typhimurium (6.6 log₁₀ CFU/ml) and increment in phage titre (3.8 log₁₀ PFU/ml) was observed. Φ st1 produced adsorption rates of 88.4-92.2% at pH7-9 and demonstrated the highest bacteria reduction (6.6 log₁₀ CFU/ml) at pH9. Φ st1 also showed an insignificant different (P>0.05) reduction rate of host cells at 37 °C (6.4 log₁₀ CFU/ml) and 42 °C (6.0 log₁₀ CFU/ml). The in vivo study using Φ st1 showed that intracloacal inoculation of ~10¹² PFU/ml of the phage in the chickens challenged with ~10¹⁰ CFU/ml of S. Typhimurium was able to reduce (P<0.05) the S. Typhimurium more rapidly than the untreated group. The Salmonella count reduced to 2.9 log₁₀ CFU/ml within 6h of post-challenge and S. Typhimurium was not detected at and after 24h of post-challenge. Reduction of Salmonella count in visceral organs was also observed at 6h post-challenge. Approximately 1.6 log₁₀ FU/ml Φ st1 was found to persist in the caecal wall of the chicks at 72 h of post-challenge. The present study indicated that Φ st1 may serve as a potential biocontrol agent to reduce the Salmonella count in caecal content of chickens.
The influenza virus is still one of the most important respiratory risks affecting humans which require effective treatments. In this case, traditional medications are of interest. HESA-A is an active natural biological compound from herbal-marine origin. Previous studies have reported that the therapeutic properties of HESA-A are able to treat psoriasis vulgaris and cancers. However, no antiviral properties have been reported.
The efficacy of bacteriophage EC1, a lytic bacteriophage, against Escherichia coli O78:K80, which causes colibacillosis in poultry, was determined in the present study. A total of 480 one-day-old birds were randomly assigned to 4 treatments groups, each with 4 pens of 30 birds. Birds from the control groups (groups I and II) received PBS (pH 7.4) or 10(10) pfu of bacteriophage EC1, respectively. Group III consisted of birds challenged with 10(8) cfu of E. coli O78:K80 and treated with 10(10) pfu of bacteriophage EC1 at 2 h postinfection, whereas birds from group IV were challenged with 10(8) cfu of E. coli O78:K80 only. All the materials were introduced into the birds by intratracheal inoculation. Based on the results of the present study, the infection was found to be less severe in the treated E. coli-challenged group. Mean total viable cell counts of E. coli identified on eosin methylene blue agar (designated EMB + E. coli) in the lungs were significantly lower in treated, E. coli-challenged birds than in untreated, E. coli-challenged birds on d 1 and 2 postinfection. The EMB + E. coli isolation frequency was also lower in treated birds; no E. coli was detectable in blood samples on any sampling day, and E. coli were isolated only in the liver, heart, and spleen of treated chickens at a ratio of 2/6, 1/6, and 3/6, respectively, at d 1 postinfection. The BW of birds from the E. coli-challenged group treated with bacteriophage EC1 were not significantly different from those of birds from both control groups but were 15.4% higher than those of the untreated, E. coli-challenged group on d 21 postinfection. The total mortality rate of birds during the 3-wk experimental period decreased from 83.3% in the untreated, E. coli-challenged birds (group IV) to 13.3% in birds treated with bacteriophage EC1 (group III). These results suggest that bacteriophage EC1 is effective in vivo and could be used to treat colibacillosis in chickens.
Feline coronaviruses (FCoVs) are found throughout the world. Infection with FCoV can result in a diverse range of signs from clinically inapparent infections to a highly fatal disease called feline infectious peritonitis (FIP). FIP is one of the most serious viral diseases of cats. While there is neither an effective vaccine, nor a curative treatment for FIP, a diagnostic protocol for FCoV would greatly assist in the management and control of the virus. Clinical findings in FIP are non-specific and not helpful in making a differential diagnosis. Haematological and biochemical abnormalities in FIP cases are also non-specific. The currently available serological tests have low specificity and sensitivity for detection of active infection and cross-react with FCoV strains of low pathogenicity, the feline enteric coronaviruses (FECV). Reverse transcriptase polymerase chain reaction (RT-PCR) has been used to detect FCoV and is rapid and sensitive, but results must be interpreted in the context of clinical findings. At present, a definitive diagnosis of FIP can be established only by histopathological examination of biopsies. This paper describes and compares diagnostic methods for FCoVs and includes a brief account of the virus biology, epidemiology, and pathogenesis.
The prevalence of feline coronavirus (FCoV) was studied in two catteries in Malaysia. Rectal swabs or faecal samples were collected from a total of 44 clinically healthy Persian purebred and mix-breed cats. RNA extracted from the faecal material was subjected to a reverse transcription-polymerase chain reaction (RT-PCR) using primers flanking for a conserved region of the virus genome. The overall prevalence of FCoV infection was 84% and the infection rate was higher in Persian purebred cats (96%) than mix-breed cats (70%). There was no significant association between the age or gender of tested cats and shedding the virus. This study is the first PCR-based survey for FCoV in Malaysia and showed the ubiquitous presence of FCoV in Malaysian cat colonies.
Among the bacterial fermentation end products in the chicken cecum, butyrate is of particular importance because of its nutritional properties for the epithelial cell and pathogen inhibitory effects in the gut. An in vitro experiment, operated with batch bioreactor, was conducted to quantify butyric-producing bacteria in a simulated broiler cecum supplemented with Lactobacillus salivarius ssp. salicinius JCM 1230 and Lactobacillus agilis JCM 1048 during 24 h of incubation. Selected bacterial species were determined by real-time PCR and short-chain fatty acids and lactate concentrations were monitored. The results showed that after 24 h of incubation, Lactobacillus supplementation significantly increased the number of lactobacilli, bifidobacteria and Faecalibacterium prausnitzii in medium containing cecal content and lactobacilli supplementation (Cc + L) compared with the control (Cc). Addition of lactobacilli did not alter Escherichia coli and Clostridium butyricum, whereas it significantly (P < 0.05) reduced Salmonella in treatment Cc + L compared with the Cc treatment. Propionate and butyrate formation were significantly (P < 0.05) increased in treatment Cc + L as compared with the Cc treatment. Lactate was only detected in treatment containing 2 Lactobacillus strains. After 24 h of incubation, acetate concentration significantly (P < 0.05) decreased in all treatments. It was suggested that lactate produced by Lactobacillus in the cecal content improved the growth of butyric producers such as F. prausnitzii, which significantly increased butyrate accumulation. Additionally, the results showed that butyrate and propionate inhibited Salmonella without influencing the E. coli profile.
Sequence analysis of the fusion (F) gene of eight Malaysian NDV isolates showed that all the isolates were categorized as velogenic viruses, with the F cleavage site motif (112)R-R-Q-K-R(116) or (112)R-R-R-K-R(116) at the C-terminus of the F(2) protein and phenylalanine (F) at residue 117 at the N-terminus of the F(1) protein. Phylogenetic analysis revealed that all of the isolates were grouped in two distinct clusters under sub-genotype VIId. The isolates were about 4.8-11.7% genetically distant from sub-genotypes VIIa, VIIb, VIIc and VIIe. When the nucleotide sequences of the eight Malaysian isolates were compared phylogenetically to those of the old published local isolates, it was found that genotype VIII, VII, II and I viruses exist in Malaysia and caused sporadic infections. It is suggested that genotype VII viruses were responsible for most of the outbreaks in recent years.
An experiment was conducted to determine the effects of combining both pleasant and unpleasant contacts with human beings on physiology and behavior of broiler chickens. Birds were subjected to the following treatments: (i) received no physical or visual contact with humans (control); (ii) from d 1 to 28, chicks were individually stroked gently for 30 s once daily (PL); (iii) from d 1 to 28, chicks were picked up individually, suspended by both legs, exposed to recorded noise, and swung gently for 15 s once daily (UNPL); (iv) from d 1 to 14 and from d 15 to 28, chicks were subjected to PL and UNPL, respectively (PL-UNPL); and (v) from d 1 to 14 and from d 15 to 28, chicks were subjected to UNPL and PL, respectively (UNPL-PL). On d 42, birds from each treatment group were road-transported for 3 h. Heat shock protein (hsp) 70 expression, plasma levels of corticosterone, serum creatine kinase concentration, heterophil/lymphocyte ratios (HLR), and tonic immobility duration were determined pre- and posttransit. There were significant (P < 0.05) duration of transportation × human contact treatment interactions for HLR and hsp 70 density. Following transit, the PL chicks had significantly (P < 0.05) lower HLR and greater hsp 70 density than the other groups. The corticosterone of PL and UNPL chicks were lower than their control, PL-UNPL, and UNPL-PL counterparts. The PL and PL-UNPL treatments were effective in shortening tonic immobility duration significantly (P < 0.05). Except for UNPL-PL, the serum creatine kinase activity of PL was significantly lower than the other groups. In conclusion, subjecting birds to pleasant human contact reduced stress and fear reactions to transportation by enhancing the ability to express hsp 70 in the brain. Unpleasant human contact had adverse effect on the birds' response to transportation. Early age pleasant experience with humans failed to negate the adverse effects of subsequent unpleasant contact.
A study was conducted to investigate the effectiveness of freeze-dried bovine pericardium (FDBP) as a biomaterial in diaphragmatic herniorrhapy in dogs. Eight adult dogs were randomly selected and divided into two equal groups. In FDBP group, a diaphragmatic defect was induced and repaired with an identical size of FDBP. In the control group, a diaphragmatic wall was incised at three-side border creating a flap and sutured. Grossly, only mild intrathoracic adhesion was observed for most of the animals, and no herniation occured. Microscopically, the biomaterial incorporated into the host's tissue by ingrowth of young muscle fiber and massive new blood vessel formation in between the fibrous tissue.
The aim of this study was to evaluate the effects of fortification and nano-size reduction on calcium absorption and bioavailability of milk powder formula in sham, ovariectomized, and ovariectomized-osteoporosis rats as a menopause and menopause-osteoporosis model. Skim milk powder and skim milk powder fortified with calcium citrate and the suitable doses of inulin, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), and vitamins D3, K1, and B6 were formulated based on the North American and Western European recommended dietary allowances. Optimization on cycle and pressure of high-pressure homogenizer was done to produce nano-fortified milk powder. In vivo study demonstrated that fortification and calcium citrate nano-fortified milk powder increased absorption and bioavailability of calcium, as well as bone stiffness and bone strength in sham, ovariectomized, and ovariectomized-osteoporosis rats. This study successfully developed an effective fortified milk powder for food application.
A study was conducted to isolate and identify chicken anaemia virus (CAV) from field samples of clinically infected broiler chickens in Malaysia. A total of 125 samples were collected from chickens aged 2-6 weeks with clinically depressed and retarded growth, of which five samples were found positive to CAV directly by polymerase chain reaction (PCR). Later, five isolates of CAV from the respective five PCR positive samples were isolated in MDCC-MSB1 cells at passage 4 based on cytopathic effects, PCR and indirect immunofluorescent antibody test. The isolates were identified as BL-1, BL-2, BL-3, BL-4 and BL-5. These CAV isolates were found to resist treatment with chloroform and heat at 37 degrees C for 2 h, 56 degrees C for 30 min and 70 degrees C for 5 min. One of the isolates, BL-5 produced significant reduction (p < 0.001) of hematocrit values (9-19%), pale bone marrow, thymus atrophy and haemorrhages in skin/muscle when inoculated into 1-day old SPF chickens. Restriction enzyme digestion of 926 bp genomic fragments of all the isolates including Cux-1 isolate with HindIII exhibited a similar pattern of bands in 2% agarose gel. The present findings confirmed the presence of CAV in Malaysia.
Two areas in the chicken anemia virus (CAV) genome have high G:C content with secondary structures. These two G:C rich areas could not be sequenced with Perkin Elmer's Big Dye Terminator Cycle Sequencing Kit. Several modifications were carried out to solve the problem. Finally, a package of modified method was developed to sequence the high G:C areas. The result showed that the Perkin Elmer's Big Dye Terminator Cycle Sequencing Kit with the normal procedures are not suitable for sequencing the high G:C regions of the CAV genome. The present developed method made the Perkin Elmer's Kit useful for the first time to sequence the G:C rich hairpin structures of the CAV genome. The system may be useful to sequence all other G:C rich DNA templates.
Base usage and dinucleotide frequency have been extensively studied in many eukaryotic organisms and bacteria, but not for viruses. In this paper, a comprehensive analysis of these aspects for infectious bursal disease virus (IBDV) was presented. The analysis of base usage indicated that all of the IBDV genes possess equivalent overall nucleotide distributions. However when the base usage at each codon positions was analysed by using cluster analysis, the VP5 open reading frame (ORF) formed a different cluster isolated from the other genes. The unusual base usage of VP5 ORF may indicate that the gene was originated by the virus "overprinting strategy", a strategy in which virus may create novel gene by utilizing the unused reading frames of its existing genes. Meanwhile, the GC content of the IBDV genes and the chicken's coding sequences was comparable; suggesting the virus imitation of the host to increase its translational efficiency. The analysis of dinucleotide frequency indicated that IBDV genome had dinucleotide bias: the frequencies of CpG and TpA were lower and the TpG was higher than the expected. Classical methylation pathway, a process where CpG converted to TpG, may explain the significant correlation between the CpG deficiency and TpG abundance. "Principal component analysis of the dinucleotide frequencies" (DF-PCA) was used to analyse the overall dinucleotide frequencies of IBDV genome. DF-PCA on the hypervariable region and polyprotein (VPX-VP4-VP3) gene showed that the very virulent IBDV (vvIBDV) was segregated from other strains; which meant vvIBDV had a unique dinucleotide pattern. In summary, the study of base usage and dinucleotide frequency had unravelled many overlooked genomic properties of the virus.