Development of in-house ELISA based on recombinant gag proteins of small ruminant lentiviruses isolated from goats in Thailand.

Small ruminant lentiviruses (SRLVs), are grouped in Retroviridae family, remain a significant loss in the small ruminant husbandry. As a result of unavailability of vaccine and effective treatment, the diagnosis plays a crucial role for the control of SRLV infection. However, the major challenge of diagnosis of SRLV infection is the genetic and antigenic variability of the viruses that can lead to a failure in serological detection. This study investigated the circulating strains of the viruses in goats in Thailand and an in-house ELISA was developed. The coding sequences for gag protein were optimized, synthesized, and expressed in Escherichia coli for increasing the sensitivity of ELISA test. A total of 365 serum samples were examined against the recombinant protein in an in-house ELISA. The results showed that the recombinant gag achieves 96.67% sensitivity and 93.18% specificity as compared with the commercially available ELISA test kit.

Development of a recombinase-aided amplification method combined with lateral flow dipstick assay to detect Porcine circovirus type 2.

Background and Aim: Porcine circovirus type 2 (PCV2) is a pathogenic virus that suppresses the immune system of pigs, impacting their health and causing economic losses. Rapid diagnostic tools for early detection of PCV2 are critical to disease prevention and control. Several molecular techniques have been established for detecting PCV2 but costly equipment and time-consuming methods are unsuitable for field inspection. In this study, we developed a recombinase-aided amplification combined with lateral flow dipstick (RAA-LFD) assay to compare with polymerase chain reaction (PCR) and quantitative PCR (qPCR) in detecting PCV2 in suspected field samples. Materials and Methods: To amplify RAA products, 15 primer pairs were designed from the conserved region of the open reading frame (ORF) 1 gene based on multiple alignments of eight PCV2 genotypes. The most efficient primer pair and conditions for the RAA-LFD assay were tested and selected. Limit of detection, repeatability, and reproducibility were determined using the constructed plasmid. DNA was extracted from positive samples for specificity testing as well as from 100 field samples to compare the detection of the RAA-LFD assay with PCR and qPCR. Results: The F1/R1 primer pair was chosen and labeled with fluorescein isothiocyanate at the 5' end of the forward primer and with biotin at the 5' end of the reverse primer. The limit of detection of the RAA-LFD assay was 10 copies/µL at 38°C for 30 min. The RAA-LFD assay was repeatable and reproducible, with no cross-reaction with PCV3, Actinobacillus pleuropneumoniae, Porcine epidemic diarrhea virus, Classical swine fever virus, Porcine reproductive and respiratory syndrome virus - North America strain (PRRSV-US) and Porcine reproductive and respiratory syndrome virus - European strain (PRRSV-EU). Based on testing with 100 samples, the developed RAA showed 100% specificity and 90.56% and 85.71% sensitivity when compared to PCR and qPCR, respectively Cohen's kappa coefficients showed a good agreement with the established techniques. Conclusion: The RAA-LFD assay targeting the ORF1 gene was highly sensitive, specific, quick, and simple to perform in the field.

Effects of Crocodile Oil (Crocodylus siamensis) on Liver Enzymes: Cytochrome P450 and Glutathione S-Transferase Activities in High-fat DietFed Rats.

Crocodile oil is a highly effective treatment for ailments ranging from skin conditions to cancer. However, the effects of the oil on liver detoxification pathways are not well studied. This study aimed to investigate the effects of crocodile oil on the detoxification enzyme activities and the mRNA expressions of cytochrome P450 1A2 (CYP1A2), cytochrome P450 2E1 (CYP2E1), and glutathione S-transferase (GST) in rats. The rats were divided into four groups (n = 7/group): rats received a standard diet (C), a high-fat diet or HFD (H), and HFD with 1 ml (HCO1) and 3 ml (HCO3) of the oil per kg body weight. Interestingly, the oil yields from this study presented alpha-linolenic acid (0.96%) at similar levels compared with fish oil. The results revealed that HFD significantly increased the activity and relative gene expression of CYP1A2 in the H group (P < 0.05), whereas 3% crocodile oil normalized the enzyme activities compared to the C group. This suggested inhibiting the HFD-induced expression of CYP1A2 mediated by the omega-3 fatty acids found in the oil. Also, crocodile oil supplementation did not reduce the activities of GST. However, the relative gene expression of GSTA1 was significantly decreased (P < 0.05) in the HCO1 and HCO3 groups compared to the H group, which might be attributed to the lower lipid peroxidation that occurred in the liver tissues. Therefore, it could be suggested that using crocodile oil could help in liver detoxification through the CYP1A2 even when offered with a HFD.

Seroprevalence of bovine coronavirus and factors associated with the serological status in dairy cattle in the western region of Thailand.

BACKGROUND AND AIMS: Bovine coronavirus (BCoV) is a pathogen affecting the productivities of dairy cattle worldwide. The present study aimed to determine the seroprevalence and factors associated with BCoV serological status using a commercial indirect enzyme-linked immunosorbent assay (ELISA). MATERIALS AND METHODS: A cross-sectional study was conducted in the western region of Thailand. Blood samples were collected from 30 dairy herds. In total, 617 blood serum samples were tested using a commercial indirect ELISA for BCoV-specific immunoglobulin G antibodies. A questionnaire was used to collect data on the factors which have been identified as risk factors for BCoV antibody detection. The age and history of diarrhea of each animal were recorded. Fisher's exact test was performed to univariately assess the association between BCoV serological status and possible risk factors. Variables with Fisher's exact test p<0.10 were then evaluated using multivariate logistic regression to identify factors associated with BCoV serological status. The Bonferroni adjustment was used for multiple comparisons of significant variables in the final multivariate logistic regression model. RESULTS: No herd was free from antibodies to BCoV. The individual seroprevalence of BCoV was 97.89% (604/617). The prevalence within herds was in the range of 45.45-100%. Cattle >3 years of age were more likely to be seropositive to BCoV compared to cattle <1 year of age (p=0.003), with the odds ratio being 81.96. Disinfecting diarrhea stools were a protective factor for being BCoV seropositive, with odds ratios of 0.08 and 0.06 compared to doing nothing (p=0.008) and to clean with water (p=0.002), respectively. CONCLUSION: BCoV seropositive dairy cattle were distributed throughout the western region of Thailand. The probability of being seropositive for BCoV increased with increasing animal age. Cleaning the contaminated stool with appropriate disinfectants should be recommended to farmers to minimize the spread of the virus.

Prevalence and genetic diversity of Haemoproteus and Plasmodium in raptors from Thailand: Data from rehabilitation center.

The diurnal raptors (Family: Accipitridae and Falconidae) are important as ecosystem bioindicators. Unfortunately, the global number of these birds has fallen, and they are close to extinction. This study reports the molecular prevalence and genetic diversity of Haemoproteus and Plasmodium in raptors admitted to the Kasetsart University Raptor Rehabilitation Unit over a period of 6 years. A total of 198 raptors, including 22 species from 30 provinces in Thailand, were admitted. The prevalence of parasites in raptors was low: Haemoproteus was 4.04% (95% CI: 1.29-6.78), and Plasmodium 2.53% (95% CI: 0.34-4.71). Eleven lineages of haemosporidian parasites were identified, and four lineages (ACCBAD02, NISALB01, NISALB02, and AEGMO03) are new globally. Interestingly, six lineages were isolated from birds belonging to the Accipitridae and Falconidae families (TYTAL4, TYTAL6, GLACUC08, MILANS06, OTUSCO02, and ORW1), indicating host shift of these parasites. Furthermore, the low prevalence of Haemoproteus and Plasmodium in raptors compared with that in previous reports suggests a relationship between the activity of avian hosts and vectors. This information is valuable for application in raptor rehabilitation and further research.

Scorpion Venom Peptide Effects on Inhibiting Proliferation and Inducing Apoptosis in Canine Mammary Gland Tumor Cell Lines.

The most common neoplasms in intact female dogs are CMGTs. BmKn-2, an antimicrobial peptide, is derived from scorpion venom and has published anticancer effects in oral and colon human cancer cell lines. Thus, it is highly likely that BmKn-2 could inhibit CMGT cell lines which has not been previously reported. This study investigated the proliferation and apoptotic properties of BmKn-2 via Bax and Bcl-2 relative gene expression in two CMGT cell lines, metastatic (CHMp-5b) and non-metastatic (CHMp-13a). The results showed that BmKn-2 inhibited proliferation and induced apoptosis in the CMGT cell lines. The cell morphology clearly changed and increased apoptosis in a dose dependent of manner. The half maximum inhibitory concentration (IC50) was 30 µg/mL for CHMp-5b cell line and 54 µg/mL for CHMp-13a cell line. The induction of apoptosis was mediated through Bcl-2 and Bax expression after BmKn-2 treatment. In conclusion, BmKn-2 inhibited proliferation and induced apoptosis in both CHMp-5b and CHMp-13a cell lines via down-regulation of Bcl-2 and up-regulation of Bax relative mRNA expression. Therefore, BmKn-2 could be feasible as candidate treatment for CMGTs.

Molecular and Morphological Analyses of Leucocytozoon Parasites (Haemosporida: Leucocytozoidae) in Raptors From Thailand.

INTRODUCTION: Leucocytozoon spp. causes a vector-borne disease that is nonpathogenic in domestic and wild birds. To date, there was no report of leucocytozoonosis in raptors from Thailand. METHODS: This study was carried out to perform morphological and molecular analyses of Leucocytozoon in 400 raptors at a rehabilitation center at Kasetsart University, Thailand during a 7-year period. The nested PCR was used to amplify the cytochrome b gene of Leucocytozoon with primers HaemNF1 and HaemNR3 as the primary reaction. RESULTS: The light microscopic examination revealed Leucocytozoon gametocytes in five raptors; three diurnal raptors [two Crested Goshawks (CGs, Accipiter trivirgatus) and one Eastern Imperial Eagle (EIE, Aquila heliaca)], and two nocturnal raptors (one Oriental Scops-Owl (OSO, Otus sunia,) and one Short-eared Owl, Asio flammeus) and two species were identified: Leucocytozoon danilewskyi in both owl species and L. californicus in two CGs. The PCR method revealed more infection rate (2.0%, 8/400) than the light microscopic method including one Barred Eagle-Owl (BEO, Bubo sumatranus), one Brown Hawk Owl (BHO, Ninox scutulata) and one OSO. A phylogeny revealed that sequences from one SEO and one OSO were clustered with L. danilewskyi and the three Leucocytozoon sequences from diurnal raptors were clustered with L. californicus. The other three sequences from a BHO, a BEO and an OSO were ambiguous. CONCLUSION: This study combined morphological, morphometric and molecular phylogenetic analyses to identify L. danilewskyi in two species of owls, L. californicus in three diurnal raptors, and unknown species in three other owls, representing the first records of leucocytozoon infection in raptors from Thailand.

Molecular studies on oestrogen α and progesterone receptors and histomorphometric analysis of canine uteri following aglepristone treatment.

Aglepristone, a competitive progesterone antagonist, is successfully used in various progesterone-dependent conditions. This study investigated uterine histomorphometric analysis, and expressions of the oestrogen α receptor (ERα) and progesterone receptor (PR) in uteri of bitches following the single dose of aglepristone treatment. Twelve client-owned healthy diestrous bitches were used in the study. The single dose of aglepristone (Alizine® , 10 mg/kg) was injected subcutaneously 5 days before ovariohysterectomy in the treatment group (n = 6); bitches without treatment served as a control group (n = 6). Uteri were collected for histomorphometric analysis, ERα and PR gene, and protein expressions studies. The mRNA expressions of ERα and PR were determined by RT-qPCR. Immunohistochemical analysis was used to evaluate the ERα and PR protein expressions using an H-score in five parts of the uterus. The results demonstrated glandular epithelium height significantly decreased (p < .05) and ERα mRNA increased (p < .01) in treated dogs. Of the treated bitches, lower expression levels of ERα were observed in the luminal epithelium, crypt and glandular epithelium, with higher expression in the endometrial stroma and myometrium (p < .05); however, PR expression decreased in the luminal epithelium, crypt and glandular epithelium (p < .01). In conclusion, reduction of the uterine glandular epithelium and ERα mRNA upregulation together with changes in ERα and PR expressions were observed in the treated bitches. However, changes in uterine ERα and PR expressions in the treated bitches depended on tissue layers. The treatment had no effect on serum oestradiol and progesterone levels.

Expression of the recombinant C-terminal of the S1 domain and N-terminal of the S2 domain of the spike protein of porcine epidemic diarrhea virus.

BACKGROUND AND AIM: Porcine epidemic diarrhea virus (PEDV) causes severe diarrhea in suckling piglets, leading to severe economic losses in the swine industry. Commercial vaccines have limited effectiveness against different genogroups of PEDV and the shedding of virus. The C-terminal of the S1 domain and the N-terminal of the S2 domain (S1-2) protein of the spike (S) protein have four neutralizing epitopes. However, research on the expression of the S1-2 segment of the S gene has been limited. In this study, we expressed a recombinant S1-2 protein of the S protein of the PEDV Thai isolate and characterized the immunological properties of the recombinant S1-2 protein. MATERIALS AND METHODS: The S1-2 segment of the S gene of the PEDV Thai isolate (G2b) was amplified, cloned into the pBAD202/D-TOPO® vector (Invitrogen, Carlsbad, CA, USA), and expressed in Escherichia coli. The optimum concentration of arabinose and the optimum induction time for the expression of the recombinant S1-2 protein were determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The immunogenic reactivity of the recombinant S1-2 protein was determined using Western blot analysis with rabbit polyclonal antibodies against the SM98 strain of PEDV (G1a). RESULTS: The recombinant S1-2 segment of the S gene of the PEDV Thai isolate protein was cloned and the recombinant S1-2 protein was successfully expressed. The optimum concentration of arabinose and the optimum induction time for the induction of the recombinant S1-2 protein were 0.2% and 8 h, respectively. The recombinant S1-2 protein reacted specifically with both rabbit anti-histidine polyclonal antibodies and rabbit anti-PEDV polyclonal antibodies. CONCLUSION: The recombinant S1-2 protein reacted with rabbit anti-PEDV polyclonal antibodies induced by the different PEDV genogroup. Therefore, the recombinant S1-2 protein may be a useful tool for the development of a diagnostic test for PEDV or for a vaccine against PEDV.

Genotypic and phenotypic situation of antimicrobial drug resistance of Escherichia coli in water and manure between biogas and non-biogas swine farms in central Thailand.

Currently, Thai livestock is rapidly expanding, especially the production of ruminants, chicken, and swine. The improper use of antibiotics will probably lead to an antimicrobial resistance problem. It has long been suspected that wastewater released from swine farms is a crucial aspect of the spread of antimicrobial resistance to the environment. Biogas systems are wastewater treatment systems commonly used on swine farms; however, little is known about the roles they play in the occurrence and transmission of resistant bacteria between biogas and non-biogas systems. This study collected pooled water, wastewater, and feces samples from five biogas farms and three non-biogas farms in Central Thailand. The samples were isolated to hemolytic E. coli (HEC) and non-hemolytic E. coli (NHEC) to test the drug resistance by using VITEK® 2 Compact (BioMérieux, USA) and detect resistant genes by using the polymerase chain reaction (PCR) technique to correlate the determined phenotypic and genotypic patterns. The results demonstrated that enumeration levels of E. coli ranged from 20.1 to 70.4 (MPN/100 ml), 105 to 107 (cfu/ml), and 105 to 109 (cfu/g), while they were 0-148.7 (MPN/100 ml), 105 to 107 (cfu/ml) and 105 to 109 (cfu/g) for water, wastewater and manure from biogas and non-biogas swine farms, respectively. The amount of E. coli in the sow feces samples was higher than the samples of nursery piglets on biogas farms at a 0.05 significant level (p < 0.05). The antimicrobial resistance indicated the relevant resistance characteristics of E. coli: the highest antimicrobial resistance was for ampicillin (AMP), followed by amoxicillin (AMX), tetracyclines (TET), chloramphenicol (C), and piperacillin (PIP), respectively. Multidrug resistance (MDR) of E. coli was 15 drugs: AMP-AMX-AMC-PIP-CEX-CEV-CPD-XNL-GM-IMP-SXT-C-TE (11.9%) and AMP-AMX-AMC-PIP-CEX-CEV-CPD-XNL-GM-IMP-SXT-C-ENR-MBR-TE (18.55%), which were the most commonly found in biogas and non-biogas swine farms, respectively. The blaTEM, tetA, sul2, and sul3 were dominantly resistant genes isolated from the water from both types of farm; while, blaTEM, aadA1, tetA, dfrA12, sul2, sul3, and cmlA were isolated from feces. The amount of E. coli in the final effluent from biogas swine farms was higher than the non-biogas swine farms; however, it was not significantly different at (p > 0.05). Furthermore, the findings of study found that genotypic characteristic of HEC showed similarity 100%. Thus, it was concluded that the levels of E. coli were accelerated in biogas wastewater treatment systems, and isolated E. coli demonstrated multidrug resistance. Even though E. coli was found in different locations, it showed relevant resistance characteristics. Therefore, regular monitoring of antimicrobial resistance on livestock farms is necessary for efficient management and drug uses on farms.

Biological and environmental factors associated with the detection of elephant endotheliotropic herpesvirus in Asian elephants (Elephas maximus) in Thailand.

Elephant endotheliotropic herpesvirus (EEHV) infection is one of the most common diseases in young elephants, causing severe fatal hemorrhagic disease. Subclinical infection was previously described; however, information about the factors associated with virus shedding and reactivation were scarce. To identify the biological and environmental factors related with EEHV detection, blood and oral swab samples were collected from nine captive Asian elephants in Thailand for one year and tested for EEHV presence using real-time PCR. Data including hematological values, management, environmental temperature, and serum cortisol levels were also recorded and analyzed. Results showed that the viral detection frequency ranged from 0-25%. The highest detection frequency was found in the two youngest elephants, aged less than 15 years. Three types of viruses, EEHV1, EEHV4, and EEHV5, were found in this study, which also detected mixed infection in five elephants. Additionally, the study found that sample type, changes in hematological values, management and health issues, and serum cortisol levels were not associated with herpesvirus detection in the elephants. However, EEHV detection percentage was significantly increased in the summer (mid-Feb to mid-May), possibly due to body fitness reduction from food source limitation and low nutrient content. To obtain a broad aspect of EEHV management, long-term EEHV monitoring is highly recommended in every captive elephant herd.

Subclinical infection of captive Asian elephants (Elephas maximus) in Thailand with elephant endotheliotropic herpesvirus.

Elephant endotheliotropic herpesvirus (EEHV) infection is a conservation threat to the endangered Asian elephant (Elephas maximus), causing fatal hemorrhagic disease in juvenile elephants throughout the world, including Thailand. This study revealed a subclinical EEHV1 infection rate of 5.5% in healthy captive Asian elephants in Thailand (n = 362). The virus was detected in all age classes above one year old, in both sexes, and across the country - even in facilities with no history of hemorrhagic disease (EEHV HD). Subclinical EEHV infection in Thailand urgently requires proper health management.

Molecular prevalence and phylogenetic relationship of Haemoproteus and Plasmodium parasites of owls in Thailand: Data from a rehabilitation centre.

Owls are nocturnal raptors that are prevalently infected with haemosporidian parasites wordwide. These birds were commonly submitted to the Kasetsart University Raptor Rehabilitation Unit, Kasetsart University, Thailand and were examined using PCR-based methods for the presence of haemosporidian infections of by the genera Plasmodium and Haemoproteus. Blood samples from 167 individual owls belonging to 12 species common in Thailand were collected between September 2012 and February 2018. The overall prevalence of haemosporidians was 34.1%, with Haemoproteus infections (25.1%) being more prevalent than Plasmodium infections (9.0%). The prevalence of both Haemoproteus and Plasmodium parasites was similar in all seasons of the year. Molecular characterization revealed 17 new haemosporidian parasite lineages (11 Haemoproteus and six Plasmodium), with genetic variation among partial cytochrome b sequences ranging from 0.0% to 3.6% in Haemoproteus lineages and 0.2%-8.8% in Plasmodium lineages. Phylogenetic analysis showed that all Haemoproteus lineages detected in owls appeared in one well-supported clade together with other parasites belonging to the Parahaemoproteus subgenus, indicating their close evolutionary relationship and common transmission modality by Culicoides biting midges. This study showes the existence of prominent non-described haemosporidian parasite diversity in Thai owls and provides baseline molecular information for further research on the genetic diversity of owl haemosporidian parasites. New DNA sequence information can be used for the diagnosis of owl infections, which have been often reported during rehabilitation planning.

Morphological and molecular characterization of avian trypanosomes in raptors from Thailand.

From September 2012 to May 2018, blood samples from 364 raptors (mostly adults) were collected and screened for trypanosomes and haemosporidians by microscopic examination and nested polymerase chain reactions (PCR). Trypanosoma spp. were identified in 15 birds from eight different species. Light microscopy revealed 14 cases of infection with Trypanosoma cf. corvi, including one each in black-shouldered kite (Elanus caeruleus, n = 49), Brahminy kite (Haliastur indus, n = 50), and spotted owlet (SO, Athene brama, n = 27); two mountain hawk-eagles (Spizaetus nipalensis, n = 3); and three each in Asian barred owlets (ABO, Glaucidium cuculoides, n = 27), barn owls (BO, Tyto alba, n = 65) and collared scops owls (CSO, Otus lettia, n = 41). In addition, one case of infection with T. avium was identified in an oriental scops owl (OSO, Otus sunia, n = 2). All infected raptors showed very low parasitemia levels. The PCR detected more three positives in one CSO, one Japanese sparrowhawk (Accipiter gularis), and one OSO. The sensitivity and specificity of the PCR method were 93.3% and 99.1%, respectively. The overall infection rate was very low (4.9%). The highest infection rate was recorded in cold-dry season (9.9%). Coinfection of Plasmodium with trypanosomes was found in all three ABOs. Coinfection with Haemoproteus spp. was found in one BO, three CSOs, and one SO. Coinfection with Haemoproteus spp. and Leucocytozoon danilewskyi was found in the OSO. Microfilarias were detected in one ABO and one CSO. The ultrastructure of trypomastigotes of T. cf. corvi in an ABO revealed fine structures. All small subunit ribosomal RNA (SSU rRNA) sequences belong to two clades: T. avium and T. corvi-culicavium complex/group. SSU rRNA gene amplification was not successful in one BO. The raptors with trypanosome infections showed normal hematological values and healthy appearance. Furthermore, this is the first report of T. avium in a nocturnal raptor from Thailand.

First detection and characterization of Psittaciform bornaviruses in naturally infected and diseased birds in Thailand.

In Thailand a proventricular dilation disease (PDD)-like syndrome commonly occurs in captive psittacine birds. The etiology, however, has been unknown to date and studies to detect parrot bornaviruses have never been performed in Southeastern Asia. Therefore, 111 psittacines (22 different species) including birds with suspected PDD based on clinical examination results (n = 65), cage mates of PDD suspected parrots without any clinical signs (n = 39) and dead birds with previous clinic suspicious for PDD (n = 7) were tested for bornaviruses using various reverse transcription polymerase chain reaction (RT-PCR) and realtime RT-PCR protocols, an enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, and genome sequencing. Bornaviral infections, indicated by the presence of RNA or antibody positive reactions were detected in 60 birds (54.1%) belonging to 15 psittaciform species and originating from 41 owners. Occurrence of Psittaciform 1 orthobornavirus was confirmed by sequencing of PCR products in 24 of these birds. Parrot bornavirus (PaBV)-5, belonging to the species Psittaciform 2 orthobornavirus and found only in single birds in the United States of America, Japan and Hungary until now, was identified in a macaw. Full genome sequencing revealed features shared with other strains of this virus. PaBV-4 was the prevalent virus type and the viruses grouped in two of the five genetic PaBV-4 subclusters known so far while PaBV-2 was found in a single patient. Forty-five psittacines of the group of PDD-suspected birds (69.2%), 4 dead birds and 11 clinically healthy cage mates were positive in at least one test the latter suggesting inefficient horizontal transmission in natural infections. Lymphoplasmacytic infiltrations (non-purulent inflammation, ganglioneuritis) and bornavirus antigen were detected in diverse tissues confirming PDD as the disease involved. These results may have a major impact on conservation projects including the five near-threatened parrot species living in the wild in Thailand.

Haemoproteus in barn and collared scops owls from Thailand.

The barn owl (BO) and the collared scops owl (CSO) are common nocturnal raptors throughout Thailand. Blood samples from 23 adult BOs and 14 CSOs were collected and processed for complete blood cell counts and parasite morphological examinations. Two Haemoproteus-positive samples were processed for ultrastructural observation. Polymerase chain reaction (PCR) analysis for a partial cytochrome b gene (cytb) from Haemoproteus was performed in all samples. Haemoproteus presence detected by light microscopy was lower than that detected by PCR (30.4% and 34.8%, respectively, in BO; and 50.0% and 78.6%, respectively, in CSO). Comparative hematology revealed that Haemoproteus-positive BOs had higher mean cell hemoglobin concentration, total leukocyte, absolute heterophil, basophil, and monocyte counts than Haemoproteus-negative BOs, but no significant differences between Haemoproteus-negative and -positive CSOs. Monocyte ultrastructure analysis revealed a role in the elimination of gametocytes. Morphologically, the Haemoproteus in 3 BOs and 6 CSOs were identified as H. noctuae, while that in 1 CSO was identified as H. syrnii. Phylogenetic analysis indicated the Haemoproteus spp. in 8 BOs and 7 CSOs were not closely related to H. noctuae or H. syrnii, and the cytb of 2 CSOs was that of H. syrnii. These results should be useful for study of Haemoproteus.

Molecular and phylogenetic characterization of bovine coronavirus virus isolated from dairy cattle in Central Region, Thailand.

Bovine coronavirus (BCoV) is involved mainly in enteric infections in cattle. This study reports the first molecular detection of BCoV in a diarrhea outbreak in dairy cows in the Central Region, Thailand. BCoV was molecularly detected from bloody diarrheic cattle feces by using nested PCR. Agarose gel electrophoresis of three diarrheic fecal samples yielded from the 25 samples desired amplicons that were 488 base pairs and sequencing substantiated that have BCoV. The sequence alignment indicated that nucleotide and amino acid sequences, the three TWD isolated in Thailand, were more quite homologous to each other (amino acid at position 39 of TWD1, TWD3 was proline, but TWD2 was serine) and closely related to OK-0514-3strain (virulent respiratory strain; RBCoV).The amino acid sequencing identities among TWD1, TWD2,TWD3, and OK-0514-3 strain were 96.0 to 96.6%, those at which T3I, H65N, D87G, H127Y, andQ136R were changed. In addition, the phylogenetic tree of the hypervariable region S1subunit spike glycoprotein BCoV gene was composed of three major clades by using the 54 sequences generated and showed that the evolutionally distance, TWD1, TWD2, and TWD3 were the isolated group together and most similar to OK-0514-3 strain (98.2 to 98.5% similarity). Further study will develop ELISA assay for serologic detection of winter dysentery disease.

Cell penetrable human scFv specific to middle domain of matrix protein-1 protects mice from lethal influenza.

A new anti-influenza remedy that can tolerate the virus antigenic variation is needed. Influenza virus matrix protein-1 (M1) is highly conserved and pivotal for the virus replication cycle: virus uncoating, assembly and budding. An agent that blocks the M1 functions should be an effective anti-influenza agent. In this study, human scFv that bound to recombinant M1 middle domain (MD) and native M1 of A/H5N1 was produced. Phage mimotope search and computerized molecular docking revealed that the scFv bound to the MD conformational epitope formed by juxtaposed helices 7 and 9 of the M1. The scFv was linked molecularly to a cell penetrable peptide, penetratin (PEN). The PEN-scFv (transbody), when used to treat the cells pre-infected with the heterologous clade/subclade A/H5N1 reduced the viral mRNA intracellularly and in the cell culture fluids. The transbody mitigated symptom severity and lung histopathology of the H5N1 infected mice and caused reduction of virus antigen in the tissues as well as extricated the animals from the lethal challenge in a dose dependent manner. The transbody specific to the M1 MD, either alone or in combination with the cognate human scFvs specific to other influenza virus proteins, should be an effective, safe and mutation tolerable anti-influenza agent.

First reported case of elephant endotheliotropic herpes virus infection in Laos.

The elephant endotheliotropic herpesvirus (EEHV) is now recognized as one of the main causes of death of young Asian elephants (Elephas maximus) in North American zoos. Its impact in wild and domestic elephant populations in Asia is not clearly understood. This article describes the first case of EEHV infection in Lao People's Democratic Republic of a 2.5-yr-old domestic male Asian elephant. Clinical signs and pathological findings reported here are consistent with previous infections in Asian elephant calves. Phylogenetic analyses showed 100% homology with other EEHV-1A strains identified in Asia, Europe, and North America. Contamination of the molecular assays was ruled out, because the DNA polymerase sequence identified in this study differed from the positive control by two base pairs.

Developing an indirect ELISA based on recombinant hexon protein for serological detection of inclusion body hepatitis in chickens.

Fowl adenovirus (FAdv) serotype 2 causes inclusion body hepatitis (IBH) disease which adversely affects the broiler industry in Thailand. We developed an indirect ELISA based on the recombinant hexon protein produced by E. coli. The recombinant hexon protein was tested with sera, in both infected and noninfected chickens. The recombinant hexon protein was standardized with an antigen concentration of 3.75 µg/ml and test sera. The intra- and inter-assays were repeatable. The cutoff value from TG-ROC curve analysis was 0.106. The specificity and sensitivity were 80 and 80%, respectively. The correlation coefficient (r) of absorbance values from this ELISA compared with the serum neutralization test was 0.76. This ELISA might be helpful for IBH diagnosis and surveillance.