The Expression Levels of CD20 as a Prognostic Value in Feline B-Cell Nasal Lymphoma: A Pilot Study.

The effect of the semi-quantitative expression of CD20 in the prognosis of feline nasal lymphoma has not been described. This study investigated the prognostic significance of CD20 expression, clinicopathological characterization, and treatment outcomes in cats with nasal lymphoma. Clinical data from cats diagnosed with nasal lymphoma were retrospectively collected, including signalment, clinical signs, clinicopathological variables, treatment outcomes, and survival times. Using ImageJ software, CD20 expression was semi-quantitatively measured based on the proportion of CD20-positive areas. Correlations between laboratory findings, immunohistochemical expressions, and survival outcomes were investigated. All cats included in the study exhibited the B-cell immunophenotype. During treatment, a reduction in PCV was noted in the cats at the second and sixth weeks (p = 0.01 and p = 0.01, respectively). The cats with low CD20 expression exhibited a significantly shorter MST (91 days; 95% CI, 41-141) than those with high CD20 expression (MST, 214 days; 95% CI, 76-351) (p = 0.01). Stage T1 cats displayed a higher MST (143 days; 95% CI, 144-172) than those in other stages > T1 (120 days, 95% CI, 71-169 days) (p = 0.04). Anemia, a common adverse effect in feline nasal lymphoma, did not impact MST. T1 clinical staging and high CD20 expression showed a trend for better MST.

Genetic structure and diversity of green turtle (Chelonia mydas) in the Gulf of Thailand.

Background and Aim: The International Union for the Conservation of Nature and Natural Resources lists the green turtle as endangered. Green turtle nesting behavior in the Gulf of Thailand has decreased to <50% of the 1995 level. The population structure of green turtles in the Gulf of Thailand has not yet been studied. This study aimed to characterize the genetic diversity of green turtles in the Gulf of Thailand based on comparisons of mitochondrial DNA (mtDNA) control region with sequences of Indo-Pacific management units (MUs) and rookeries, to investigate population structures, and to explore phylogeographic relationships. Materials and Methods: Blood samples (1 mL each) from 91 stranded green turtles were collected from four parts of the Gulf of Thailand (eastern, upper, central, and lower). The control mtDNA region was amplified by polymerase chain reaction using LCM15382 and H950 primer. The obtained 384-bp or 770-bp sequences were analyzed for haplotype, clade, and haplotype and nucleotide diversities and were used to construct a phylogenetic tree and haplotype network diagram, respectively. In addition, we analyzed genetic differentiation within and among populations of green turtles in the Gulf of Thailand and between green turtles in the Gulf of Thailand and other Indo-Pacific MUs and rookeries. Results: In total, 12 (based on 384 bp) or 13 (based on 770 bp) haplotypes and two clades (clades VII and VIII) were identified, with nine or 10 haplotypes belonging to clade VIII and three haplotypes belonging to clade VII. Of the new haplotypes, four or five were identified and classified as clade VII (two haplotypes, for both fragment lengths) and clade VIII (two or three haplotypes, for 384 bp or 770 bp fragments, respectively). The overall haplotype and nucleotide diversity of green turtles in the Gulf of Thailand were high (0.755 ± 0.039 and 0.01146 ± 0.00248, respectively). Based on the analysis of molecular variance, green turtles in the Gulf of Thailand could be divided into two subpopulations (UC-Eastern Gulf of Thailand [UC-EGT] and lower Gulf of Thailand [LGT]). Comparisons with other MUs and rookeries in the Indo-Pacific showed that UC-EGT was not genetically different from the Peninsular Malaysia and Eastern Taiwan (Lanyu) MUs and the Terrangganu and Mersing rookeries, and LGT were not genetically different from Peninsular Malaysia, Sipadan, Brunei Bay, Eastern Taiwan (Lanyu), Scott Reef and Browse Island, and Gulf of Carpentaria MUs and the Perak, Perhentain Island, Redang, Pahang, and Vietnam rookeries. Conclusion: To the best of our knowledge, this is the first report to identify the haplotypes and clades of green turtles in the Gulf of Thailand and to show that the populations in the Gulf of Thailand not only present high genetic diversity but also have haplotypic endemism. Longer mtDNA fragments (770 bp) increased the resolution of the stock structure. Clade VII is a unique clade not only for Japan but also for Thailand and Malaysia, and CmP82 is a unique haplotype for both the Gulf of Thailand and Malaysia. Conservation and management of these populations are important to preserve the genetic diversity, biological diversity, and evolutionary potential of green turtles in the Gulf of Thailand.

Radiotherapy in combination with exenteration and partial orbitectomy for orbital multilobular tumor of bone in a Cocker Spaniel.

Background: Multilobular tumor of bone or multilobular osteochondrosarcoma is a tumor of flat bone in the skull. The treatment of choice for a multilobular tumor of bone is local aggressive surgical excision. Case Description: A female Cocker Spaniel dog aged 11 years presented with a history of globe displacement of the right eye for 3 months. Ophthalmic examination revealed exophthalmos, third eyelid protrusion, and slightly increased intraocular pressure OD (oculus dexter; right eye). Computed tomography (CT) revealed a mass effect in the right retrobulbar, maxilla, zygomatic, and temporal areas. Right zygomatic and temporal bone lysis were observed. Physical examination, hematology, and blood chemistry results were within normal limits. Exenteration with zygomatic arch removal was performed. During surgery, a firm 2-lobed mass (4.8 × 3.7 and 1.6 × 1.4 cm) adhered to the mandible was found in the retrobulbar area OD. Histopathological findings revealed a multilobular tumor of bone. CT imaging was performed for the remaining tumor and an extended part of the right retrobulbar mass was found. Hypofractioned radiotherapy with 6 fractions of 6 Gy was performed on days 0, 7, 14, 21, 28, and 35. At 1-month and 4-month follow-up inspections, the mass gradually reduced in size. At 8 months and 11 months after radiotherapy, the mass was unremarkable. The dog was alert during all follow-up periods to 1 year and 8 months after hypofractioned radiotherapy combined with exenteration and partial orbitectomy. Conclusion: Hypofractioned radiotherapy combined with exenteration and partial orbitectomy extended the patient's survival and decreased the size of the remaining tumor for the management of orbital multilobular tumor of bone in this dog for at least 1 year and 8 months.

Study of trace elements in stranded green turtles (Chelonia mydas), hawksbill turtles (Eretmochelys imbricata), and olive ridley turtles (Lepidochelys olivacea) in Gulf of Thailand and Andaman Sea.

The purpose of this study was to survey and compare the amounts of elements in the serum of stranded sea turtles from the Gulf of Thailand and the Andaman Sea. The sea turtles from the Gulf of Thailand had Ca, Mg, P, S, Se, and Si concentrations significantly higher than those in sea turtles from the Andaman Sea. The Ni and Pb concentrations of sea turtles from the Gulf of Thailand was higher, but not significantly so, than in sea turtles from the Andaman Sea. Rb was detected only in sea turtles from the Gulf of Thailand. This may have been related to the industrial activities in Eastern Thailand. The concentration of Br in the sea turtles from the Andaman Sea were significantly higher than those in sea turtles from the Gulf of Thailand. The higher serum concentration of Cu in hawksbill (H) and olive ridley turtles (O) than in green turtles may be due to hemocyanin, as an important component in the blood of crustaceans. The higher Fe concentration in the serum from green turtles than for H and O may be due to chlorophyll, which is an important component of chloroplasts in eel grass. Co was not found in the serum of green turtles but was found in the serum of H and O. The monitoring of important elements in sea turtles may be used as a tool to assess the levels of pollution in marine ecosystems.

First report on clinical aspects, blood profiles, bacterial isolation, antimicrobial susceptibility, and histopathology in canine pyometra in Thailand.

Background and Aim: Canine pyometra, either the closed (closed pyometra [CP]) or open (open pyometra [OP]) cervix type, is a frequent uterine disease in intact old age bitches. Therefore, early diagnosis and appropriate medical and surgical treatments are crucial to avoid the life-threatening condition in these bitches. This study aimed to investigate clinical alterations, blood parameters, causative bacteria, antimicrobial susceptibility, and uterine histopathology obtained during aseptic surgical treatment on bitches with pyometra. Materials and Methods: Sixty bitches of various breeds and ages with presumptive pyometra diagnoses were included in the study. The diagnoses were based on history, clinical examination, blood parameters, radiography, and ultrasonography. All pyometra bitches were ovariohysterectomized as an emergency surgical treatment. In addition, uterine content and tissues were submitted for bacterial isolation, antimicrobial susceptibility, and uterine histopathological analysis. Results: Except for abdominal CP distention, no specific clinical signs were linked to the pyometra type. The mean values of total white blood cell count (WBC) and plasma protein were predominantly raised in pyometra bitches regarding hematological parameters. Leukocytosis was found in both types; however, the WBC in CP was markedly higher than in OP. The mean value of blood urea nitrogen increased in the CP group. Klebsiella pneumoniae and Escherichia coli were the most frequent causative bacteria isolated in CP and OP, respectively. All isolated bacteria were 100% susceptible to imipenem, meropenem, and carbapenem. Marbofloxacin was the second most effective drug against isolated bacteria from both groups. Uncomplicated cystic endometrial hyperplasia (CEH) was not presented in the CP group. CEH and chronic endometritis (type IV), the most severe uterine histopathological changes, were discovered in the CP and OP. Conclusion: The CP and OP groups presented leukocytosis, increased plasma protein, and CEH and chronic endometritis. Depression, abdominal distention, and enlarged uterine size were the major characteristics of the CP group. Furthermore, abdominal distension is presented in other abnormalities in clinical practices, providing a differential diagnosis. Drugs in the carbapenem group were the most effective against isolated bacteria; however, they are not routinely used due to bacterial resistance concerns. Thus, marbofloxacin was recommended as an alternative medical treatment because it is convenient to manage by both oral and injection routes.

Association of bacterial isolates and antimicrobial susceptibility between prostatic fluid and urine samples in canine prostatitis with concurrent cystitis.

Most old, intact male dogs usually have prostate disorders, especially benign prostatic hypertrophy and prostatitis with or without abscesses, and concurrent cystitis. The successful treatment of dogs with prostatitis concurrent with cystitis has relied on choosing an appropriate antimicrobial drug based on a bacterial culture and drug sensitivity testing. The objective of the study was to compare the prevalence of bacterial species and results of drug susceptibility testing of bacteria that were isolated from the prostatic fluids and urine samples that were collected from dogs with both prostatitis and cystitis. One hundred and sixty intact male dogs, who presented with both diseases, were recruited for the study. The disease diagnoses were based on clinical history notes, physical examinations, abdominal ultrasonography, prostatic fluid cytology, urinalysis and bacterial cultures from both prostatic fluid and urine samples. The bacterial culture results demonstrated that the major species that were detected in either the prostatic fluid or urine samples were Staphylococcus spp., Escherichia coli, Pseudomonas spp., Streptococcus spp., Proteus mirabilis and Klebsiella pneumoniae. Staphylococcus spp. (26.5 %, 43/162) and Escherichia coli (26.1 %, 12/46) were the most prevalent species from the prostatic fluid and urine samples, respectively. Statistical tests revealed that there were no significantly different prevalence levels among the isolated bacteria between the prostatic fluid and urine samples. Imipenem and gentamicin were the most potent antimicrobial drugs tested against the bacterial isolates in the present study. However, the administration of imipenem to treat prostatitis and cystitis in dogs was of concern. Interestingly, there were no significant differences in the antimicrobial drug susceptibility trends between the prostatic fluid and urine samples. Based on these results, a urine sample might be considered as an optional sample for bacterial cultures and antimicrobial drug susceptibility testing when it is not possible to collect a prostatic fluid sample.

Expression of recombinant 35 kDa fragment of VP2 protein of canine parvovirus using Escherichia coli expression system.

BACKGROUND AND AIM: Canine parvovirus (CPV) is one of the most common viral infections in dogs, causing acute hemorrhagic gastroenteritis and high mortality. Vaccination effectively prevents CPV infection. However, the currently available CPV vaccines have concerns such as maternal immunity interference, shedding of virus vaccine, and false-positive result based on polymerase chain reaction after vaccination. A subunit vaccine can overcome these problems. This study aimed to express the recombinant 35 kDa fragment of the VP2 protein (consisting of epitopes 1-7) and the recombinant full-length VP2 protein (consisting of epitopes 1-10) and to study the ability of these two recombinant proteins to react with rabbit anti-CPV polyclonal antibodies. MATERIALS AND METHODS: The full length and 35 kDa fragment of VP2 gene of CPV were cloned into the pBAD202 Directional TOPO™ expression vector and expressed in E. coli. The recombinant full-length and the recombinant 35 kDa fragment proteins of VP2 were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. RESULTS: The recombinant full-length and the recombinant 35 kDa fragment VP2 genes were successfully cloned and expressed. The optimum concentrations of arabinose and induction time for the recombinant full-length and the recombinant 35 kDa fragment VP2 proteins were 0.2% for 6 h and 0.02% for 6 h, respectively. The recombinant full-length and the recombinant 35 kDa fragment VP2 protein molecular weights were approximately 81 and 51 kDa, respectively. The recombinant full-length and the recombinant 35 kDa fragment VP2 proteins specifically interacted with rabbit anti-CPV polyclonal antibodies. CONCLUSION: These results suggest that the recombinant 35 kDa fragment and the recombinant full-length VP2 proteins may be useful in developing a CPV diagnostic test or vaccine.

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.

Investigation of hemotropic Mycoplasma spp. genotypes in client-owned cats in Thailand.

The genetic information for three feline hemoplasmas is limited in Southeast Asia. According to the limited genetic data, this study modified a nested-PCR method targeting the 16S rRNA gene by designing a novel primary forward degenerate primer. Two hundred and thirty-one archived DNA extracts from the blood of client-owned cats with a variety of diseases were used. The modified nested PCR detected feline hemoplasma DNA in 64 of 231 (27.7 %) samples. Sanger DNA sequencing, BLAST, and phylogenetic analyses revealed nine nucleotide sequences of Mycoplasma haemofelis (Mhf) (3.9 %, 9/231), fifty-three nucleotide sequences of Candidatus Mycoplasma haemominutum (CMhm) (22.94 %, 53/231) and two nucleotide sequences of Candidatus Mycoplasma turicensis (CMtc) (0.86 %, 2/231). The phylogenetic analysis demonstrated separate genotypes of 30 DNA sequences of Thai CMhm. In addition, this analysis elucidated distinct genotypes of CMhm in Thai fishing cats (Prionailurus viverrinus). The domestic cat and Thai fishing cat groups were the two major groups separating Thai CMhm genotypes based on the 16S rRNA. One CMhm sequence in Thai fishing cats was also present in domestic cat CMhm genotypes. This result suggests that transmission of CMhm between domestic cats and Thai fishing cats has likely occurred. One Mhf sequence had low genetic identity (82 % similarity). The phylogenetic analysis confirmed that this sequence was still very closely related to Mhf reference sequences. This Mhf-like genotype could be a candidate novel Mhf genotype. This new genetic information for feline hemotropic Mycoplasma provides valuable information for future feline-related clinical studies.

Detection of specific IgM and IgG antibodies in acute canine monocytic ehrlichiosis that recognize recombinant gp36 antigens.

The efficacy of antibody detection tools for all stages of Ehrlichia canis infections and for various genotypes remains unclear. We produced recombinant gp36 (rgp36) antigens from different isolates of Thai E. canis to confirm the immunoreactivities to these recombinant proteins from naturally infected dogs. Sera and blood samples were taken from 21 dogs naturally infected with E. canis and in the clinical stages of acute phase ehrlichiosis. The expression vectors and competent E. coli produced two isolates of rgp36. These two major rgp36s were recognized by the dogs' sera in Western blotting, with both anti-dog IgM and IgG used as secondary antibodies. The two different genotypes of these local recombinant immunoreactive proteins were gp36 subgroup A (isolate 1055) and subgroup B (isolate 533). The Western blot analyses successfully identified both specific IgM and IgG from the dogs' sera. Of all 21 cases, five dogs presented specific IgM, twenty dogs presented specific IgG, and the commercial test used found fifteen seropositive dogs. There were four dogs that presented both specific IgM and IgG. Only one dog presented specific IgM only. This report is the first identification of a specific IgM in dogs in response to acute infections with E. canis. The recombinant gp36 isolates may be useful as potential antigenic material for subsequent serological tests that have a high possibility for differentiating between acute, chronic, primary, and nonprimary infections with E. canis.

Feline panleukopenia virus as the cause of diarrhea in a banded linsang (Prionodon linsang) in Thailand.

A banded linsang (Prionodon linsang) presented at our hospital with clinical signs of acute diarrhea. Fecal samples were positive for canine parvovirus (CPV) as determined by polymerase chain reaction with primers specific for both CPV and feline panleukopenia virus (FPV). The full-length VP2 was cloned, sequenced, and compared with sequences of FPV and CPV strains reported in GenBank. The amino acids that determined the host range were similar to those of FPV. Moreover, amino acid analysis of VP2 revealed over 98% homology to FPV. The FPV isolate was closely related with FPV isolates from Japan, South Korea, and China. To the best of our knowledge, this is the first study to report that banded linsang can be infected with FPV.

Association of Ehrlichia canis, Hemotropic Mycoplasma spp. and Anaplasma platys and severe anemia in dogs in Thailand.

Canine tick-borne bacteria; Ehrlichia canis, hemotropic Mycoplasma spp. and Anaplasma spp., are organisms transmitted by Rhipicephalus sanguineus ticks. However, only a few clinical studies evaluating dogs infected with these organisms and anemia condition have been published. In this study, the potential tick-borne bacteria linked to anemia were investigated in eighty-one blood samples selected from anemic dogs using a broad range nested-PCR of the 16S rRNA gene. Positive results were shown in 12/81 blood specimens (14.81%). Nucleotide sequences from the PCR products were analyzed using BLAST and resulted in identification of Ehrlichia canis (8), Candidatus Mycoplasma haematoparvum (1) and Anaplasma platys (3). Two other PCR assays were used to detect and identify the positive results of these pathogens including a specific PCR for Ehrlichia canis (gp36) and a specific nested-PCR for hemoplasma species (16S rRNA) and the phylogenetic analyses of E. canis and canine hemoplasmas were performed using these two loci. These specific PCRs revealed co-infection of E. canis and Mycoplasma haemocanis in two cases. These two male dogs had presented with jaundice, severe hemolytic anemia, severe thrombocytopenia, leukocytosis, mild azotemia and hepatitis. Ehrlichia canis was detected in a significantly greater number of severe anemia cases (PCV<15%) than moderate or mild anemia cases (PCV 16-29%) (P<0.05) and these severe anemia cases were 7-fold more at risk of having E. canis infections (odds ratio: 7.11, p=0.020). However, no statistical differences were detected between E. canis detection and degrees of thrombocytopenia or leukopenia. From the results of this study, we conclude that the severity of anemia is associated with E. canis infections rather than the severity of thrombocytopenia.

Optimization of one-step real-time reverse transcription-polymerase chain reaction assays for norovirus detection and molecular epidemiology of noroviruses in Thailand.

Noroviruses (NoVs) are an important human pathogen associated with acute viral gastroenteritis worldwide. NoVs display a significant amount of genetic heterogeneity, making it difficult to develop comprehensive detection assays. In this study, primer sets and probes were designed for a TaqMan(®)-based real-time reverse transcription-polymerase chain reaction (RT-PCR) for norovirus detection purposes. The assay was optimized and utilized as a multiplex real-time RT-PCR assay for genogroup I (GI) detection, and a singleplex real-time RT-PCR assay for genogroup II (GII) detection. The assays showed high specificity for NoV detection and no cross-reactivity was observed between GI and GII. The detection limit of the assay was as low as 10 and 50 RNA copies per reaction for GI and GII, respectively. The optimized protocol was employed to assess the presence of NoV strains in clinical samples collected throughout Thailand during December 2005 to November 2006. The percentage of NoV infections among children with acute gastroenteritis (case) was 23.8% (119/500) and for children without acute gastroenteritis (control) it was 6.8% (30/441). The frequency of NoV infections varied geographically, with the highest frequency observed in the central region and the lowest frequency in the northern region (P>0.0001). Of the 149 positive case and control specimens, GII was found to be the predominant genogroup (98.6%). Partial capsid sequences were successfully obtained from 67 NoV-positive specimens and a phylogenetic analysis was performed to genotype the viral strains. GII.4 was the most common genotype detected.

Isolation of a gene encoding a cellulolytic enzyme from swamp buffalo rumen metagenomes and its cloning and expression in Escherichia coli.

Ruminants are capable of hydrolyzing lignocellulosic residues to absorbable sugars by virtue of the microbial communities residing in their rumen. However, large sections of such microbial communities are not yet culturable using conventional laboratory techniques. Therefore in the present study, the metagenomic DNA of swamp buffalo (Bubalus bubalis) rumen contents was explored using culture-independent techniques. The consensus regions of glycosyl hydrolase 5 (GH5) family of cellulases were used as primers for PCR amplification. A full-length metagenomic cellulase gene, Umcel5B29, with a complete open reading frame (ORF) of 1611 bp was identified. The similarity search analysis revealed that Umcel5B29 is closely related to the cellulases (73% to 98% similarity) of ruminal unculturable microorganisms, indicating its phylogenetic origin. Further analysis indicated that Umcel5B29 does not contain a carbohydrate binding module (CBM). Subsequently, Umcel5B29 was overexpressed in Escherichia coli. The recombinant enzyme worked optimally at pH 5.5 and 45°C, a condition similar to the buffalo's rumen. However, the enzyme retained more than 70% of its maximal activity after incubation at pH 4-7 and more than 50% maximal activity after incubation at 30-60°C for 30 min. These characteristics render Umcel5B29 as a potential candidate for the bio-stoning process of denim.

Molecular characterization of Hepatozoon species in a Leopard Cat (Prionailurus bengalensis) from Thailand.

Hepatozoon gamonts were observed by light microscopy in neutrophils of a male, wild-caught Leopard Cat. Complete blood counts at presentation and 6 months later were unremarkable. Serologic tests were negative for both FIV and FeLV. A partial sequence of the 18S rRNA gene from the Hepatozoon found in the cat indicated that, compared with all species examined, the protozoan had the closest relationship (99.2% sequence similarity) with the Hepatozoon of the water python (Stegonotus cucullatus). The cat was clinically healthy at last report. Although Hepatozoon has been found in another wild cat in Thailand, this is the first report in a Leopard Cat. The pathogenicity of Hepatozoon in these cats remains uncertain.

Sequence analysis of VP2 gene of canine parvovirus isolates in Thailand.

Canine parvovirus (CPV) causes a very severe enteric disease especially in puppies. Twenty-six isolates of CPV were obtained from dogs at the Animal Hospital, Kasetsart University, Thailand. Whole VP2 gene of 26 isolates was amplified using polymerase chain reaction (PCR) and its sequences were analyzed. Nineteen out of 26 isolates were characterized as CPV type 2a variants and the rest of the isolates were characterized as CPV type 2b. These results indicated that both types are currently prevalent field CPV circulating in Thailand and type 2a is the predominant genotype. Neither CPV type 2 nor type 2c was observed in this study.

Avian and human influenza A virus receptors in trachea and lung of animals.

BACKGROUND: Influenza A viruses are capable of crossing the specific barrier between human beings and animals resulting in interspecies transmission. The important factor of potential infectivity of influenza A viruses is the suitability of the receptor binding site of the host and viruses. The affinities of avian and human influenza virus to bind with the receptors and the distributions of receptors in animals are different. OBJECTIVE: This study aims to investigate the anatomical distribution of avian and human influenza virus receptors using the double staining lectin histochemistry method. METHODS: Double staining of lectin histochemistry was performed to identify both SA alpha2,3 Gal and SA alpha2,6 Gal receptors in trachea and lung tissue of dogs, cats, tigers, ferret, pigs, ducks and chickens. RESULTS: We have demonstrated that avian and human influenza virus receptors were abundantly present in trachea, bronchus and bronchiole, but in alveoli of dogs, cats and tigers showed SA alpha2,6 Gal only. Furthermore, endothelial cells in lung tissues showed presence of SA alpha2,3 Gal. CONCLUSION: The positive sites of both receptors in respiratory tract, especially in the trachea, suggest that all mammalian species studied can be infected with avian influenza virus. These findings suggested that dogs and cats in close contact with humans should be of greater concern as an intermediate host for avian influenza A in which there is the potential for viral adaptation and reassortment.

Efficient amplification of light and heavy chain variable regions and construction of a non-immune phage scFv library.

Non-immune phage scFv library is one of the most attractive resources for therapeutics, diagnostics and basic research. As a matter of fact, quality of the library is limited by inefficient PCR cloning of antibody genes using degenerated primers. PCR using this type of primers is difficult to optimize conditions for efficient amplification, and therefore causes loss of antibody diversities. To overcome this problem, we described a novel two-step amplification of V(kappa) and V(H) genes with newly designed primer sets. Initially, we amplified V(kappa) and V(H) genes from their signal sequences to the joining region to keep antibody diversity as large as possible. Thereafter, highly degenerated primers were used to amplify the V(kappa) and V(H) genes from the framework region 1 to the joining region. The V(kappa) and V(H) genes from the second PCR then were linked by PCR overlapping extension to generate the scFv library. Fifteen clones from the library were randomly picked and sequenced, and the diversity of full-length scFvs was confirmed. Expression capability of clones in the library was 80% after confirmation using colony hybridization. The results demonstrated the efficiency of this strategy and the primer sets for construction of the scFv library.

Hematologic, cytochemical, ultrastructural, and molecular findings of Hepatozoon-infected flat-headed cats (Prionailurus planiceps).

BACKGROUND: The flat-headed cat (Prionailurus planiceps) is a small wild cat of Southeast Asia and is considered extremely endangered. Little is known about the hematologic values, blood cell morphology, or hemoparasites of this species in relation to other Felidae. OBJECTIVES: The objective of this study was to report basic hematologic values and describe the light microscopic, cytochemical, and ultrastructural characteristics of blood cells in 2 wild-caught flat-headed cats. In addition, molecular analysis was done of a Hepatozoon organism found in the neutrophils of both cats. METHODS: Blood samples were collected into EDTA from the cephalic vein. A CBC, manual differential count, manual reticulocyte count, cytochemical stains (Sudan black B [SBB], alpha-naphthyl acetate esterase [ANAE], and beta-glucuronidase), and scanning and transmission electron microscopy were done using standard methods. RESULTS: HCT was slightly lower and reticulocyte counts and red cell distribution width were higher than the expected values for other species of cats. Hepatozoon organisms were found in the cytoplasm of neutrophils in both cats, but the number of infected neutrophils was very low (1%-2%). Neutrophils stained strongly positive for SBB, but were negative for ANAE and beta-glucuronidase. Hepatozoon-infected neutrophils were negative for SBB, but focally positive for ANAE and beta-glucuronidase. By transmission electron microscopy, gamonts of Hepatozoon sp were observed in neutrophils, and rarely free in plasma. Infected neutrophils had fewer specific granules and more mitochondria compared with noninfected neutrophils. PCR products of partial 18S rRNA revealed that the isolate of Hepatozoon in the flat-headed cats was closely related to that of the frog Hepatozoon sp. CONCLUSIONS: These results add to our understanding of hematologic values and blood cell morphology in Hepatozoon-infected flat-headed cats as well as the molecular analysis of the Hepatozoon organism, and may be useful for the health management and evaluation of hemoparasitic disease in this species.

Identification of the Actinobacillus pleuropneumoniae serotype using PCR based-apx genes.

Actinobacillus pleuropneumoniae (A. pleuropneumoniae) is an important swine respiratory disease pathogen, which has at least 15 serotypes. There are several techniques for the serotyping of A. pleuropneumoniae, however, these techniques are time consuming. In this study, the polymerase chain reaction (PCR) technique was developed for serotyping A. pleuropneumoniae using a set of specific primer designated for the apxI, apxII, apxIII and apxIVA genes. By this PCR typing system, 10 out of the 13 reference strains of A. pleuropneumoniae were differentiated. However, it was not possible to distinguish serotype 2 from 8, serotype 5a from 5b and serotype 9 from 11. Each serotype of A. pleuropneumoniae showed its own products patterns. The PCR typing system was further applied for typing the field isolates of A. pleuropneumoniae and compared to that using the gel immunodiffusion (GID) technique. The results from both PCR and GID techniques were in accordance. Thus, the PCR typing system may provide a rapid and useful tool for typing the serotypes of A. pleuropneumoniae.