Questioning inbreeding: Could outbreeding affect productivity in the North African catfish in Thailand?

The North African catfish (Clarias gariepinus) is a significant species in aquaculture, which is crucial for ensuring food and nutrition security. Their high adaptability to diverse environments has led to an increase in the number of farms that are available for their production. However, long-term closed breeding adversely affects their reproductive performance, leading to a decrease in production efficiency. This is possibly caused by inbreeding depression. To investigate the root cause of this issue, the genetic diversity of captive North African catfish populations was assessed in this study. Microsatellite genotyping and mitochondrial DNA D-loop sequencing were applied to 136 catfish specimens, collected from three populations captured for breeding in Thailand. Interestingly, extremely low inbreeding coefficients were obtained within each population, and distinct genetic diversity was observed among the three populations, indicating that their genetic origins are markedly different. This suggests that outbreeding depression by genetic admixture among currently captured populations of different origins may account for the low productivity of the North African catfish in Thailand. Genetic improvement of the North African catfish populations is required by introducing new populations whose origins are clearly known. This strategy should be systematically integrated into breeding programs to establish an ideal founder stock for selective breeding.

Weak purifying selection in allelic diversity of the ADSL gene in indigenous and local chicken breeds and red junglefowl in Thailand.

High levels of purine and uric acid, which are associated with health issues such as gout and cardiovascular disease, are found in the meat of fast-growing broiler chickens, which raises concerns about the quality of chicken meat and the health of the consumers who consume it. High genetic homogeneity and uniformity, particularly in genes involved in the synthesis of inosine monophosphate (IMP) and subsequent process of purine synthesis, which are associated with the meat quality, are exhibited in commercial broiler chickens owing to intensive inbreeding programs. Adenosine succinate lyase (ADSL) is a key enzyme involved in de novo purine biosynthetic pathway and its genetic polymorphisms affect IMP metabolism and purine content. In this study, we investigated the polymorphism of the ADSL gene in indigenous and local chicken breeds and red junglefowl in Thailand, using metabarcoding and genetic diversity analyses. Five alleles with 73 single nucleotide polymorphisms in exon 2, including missense and silent mutations, which may act on the synthesis efficiency of IMP and purine. Their protein structures revealed changes in amino acid composition that may affect ADSL enzyme activity. Weak purifying selection in these ADSL alleles was observed in the chicken population studied, implying that the variants have minor fitness impacts and a greater probability of fixation of beneficial mutations than strong purifying selection. A potential selective sweep was observed in Mae Hong Son chickens, whose purine content was lower than that in other breeds. This suggests a potential correlation between variations of the ADSL gene and reduced purine content and an impact of ADSL expression on the quality of chicken meat. However, further studies are required to validate its potential availability as a genetic marker for selecting useful traits that are beneficial to human health and well-being.

Optimizing Bangkaew dog breed identification using DNA technology.

BACKGROUND: The Bangkaew dog is an indigenous dog breed in the Phitsanulok province of Thailand. This breed is recognized by the Fédération Cynologique Internationale (FCI), a global canine organization. The unique traits of the Bangkaew breed lead to purebred selection for breeding, while only their traits and pedigree from parental history are recorded. Determination of the risk of inbreeding depression and the origin of unknown DNA profiles is essential due to the challenges in predicting puppy characteristics, which are crucial for breed management and conservation. OBJECTIVE: This study aimed to emphasize that current allelic frequency data for the Bangkaew dog breed must be considered for precise individual identification. METHODS: Approximately 82 Bangkaew dogs from various Thai localities were studied using 15 microsatellite markers for genotypic monitoring and individual identification. Maternal genetic inheritance was assessed via mtDNA D-loop analysis. RESULTS: The results revealed high genetic diversity in the Bangkaew breed, indicating low potential for inbreeding. We also found that using a 15 loci microsatellite panel was effective for the identification of Bangkaew dogs. The optimized 10 loci microsatellite genotyping panel developed in this study presents improved identification testing efficiency, promoting both time- and cost-effectiveness. CONCLUSION: Analysis of microsatellite DNA markers in Bangkaew dogs using an optimized panel of 10 loci selected from 15 loci effectively facilitated individual identification. This approach not only enhances time and cost efficiency, but also provides accurate allelic frequency estimates, which are crucial for the realistic evaluation of DNA evidence.

Overcoming taxonomic challenges in DNA barcoding for improvement of identification and preservation of clariid catfish species.

DNA barcoding without assessing reliability and validity causes taxonomic errors of species identification, which is responsible for disruptions of their conservation and aquaculture industry. Although DNA barcoding facilitates molecular identification and phylogenetic analysis of species, its availability in clariid catfish lineage remains uncertain. In this study, DNA barcoding was developed and validated for clariid catfish. 2,970 barcode sequences from mitochondrial cytochrome c oxidase I (COI) and cytochrome b (Cytb) genes and D-loop sequences were analyzed for 37 clariid catfish species. The highest intraspecific nearest neighbor distances were 85.47%, 98.03%, and 89.10% for COI, Cytb, and D-loop sequences, respectively. This suggests that the Cytb gene is the most appropriate for identifying clariid catfish and can serve as a standard region for DNA barcoding. A positive barcoding gap between interspecific and intraspecific sequence divergence was observed in the Cytb dataset but not in the COI and D-loop datasets. Intraspecific variation was typically less than 4.4%, whereas interspecific variation was generally more than 66.9%. However, a species complex was detected in walking catfish and significant intraspecific sequence divergence was observed in North African catfish. These findings suggest the need to focus on developing a DNA barcoding system for classifying clariid catfish properly and to validate its efficacy for a wider range of clariid catfish. With an enriched database of multiple sequences from a target species and its genus, species identification can be more accurate and biodiversity assessment of the species can be facilitated.

Genetic admixture and diversity in Thai domestic chickens revealed through analysis of Lao Pa Koi fighting cocks.

Lao Pa Koi (LPK) chicken is a popular fighting breed in Thailand, prized for (its unique characteristics acquired by selective breeding), and a valuable model for exploring the genetic diversity and admixture of red junglefowls and domestic chickens. In this study, genetic structure and diversity of LPK chicken were assessed using 28 microsatellite markers and mitochondrial DNA (mtDNA) D-loop sequences, and the findings were compared to a gene pool library from "The Siam Chicken Bioresource Project". High genetic variability was observed in LPK chickens using mtDNA D-loop haplotype analysis, and six haplotypes were identified. Microsatellite data revealed 182 alleles, with an average of 6.5 alleles per locus. These results confirmed the occurrence of genetic admixture of red junglefowl and Thai domestic chickens in LPK chicken breed. A maximum entropy modeling approach was used to analyze the spatial suitability and to assess the adaptive evolution of LPK chickens in diverse local environments. The model identified 82.52% of the area studied as unsuitable, and 9.34%, 7.11%, and 2.02% of the area indicated moderate, low, and high suitability, respectively. The highest contribution rate to land suitability for LPK chickens was found at an elevation of 100-250 m, suggesting the importance of elevation for their potential distribution. The results of this study provide valuable insights into the genetic origin of LPK chicken breed and identify resources for future genetic improvement.

Molecular detection of Loxodontofilaria spp. in Asian elephants (Elephas maximus) from elephant training camps in Thailand.

Filarial infection is an important disease in human and animal medicine. Several filarial worms are of importance, especially nematodes in the Onchocercidae. The Asian elephant (Elephas maximus) is an endangered animal and is very important from several socio-economic and ecological aspects in Thailand. Various parasites can be found in elephants; however, data related to filarial infections in elephants is limited. The objective of this study was to detect filaria in the blood of Asian elephants in Thailand, based on a polymerase chain reaction (PCR) technique. Blood samples were collected from 208 Asian elephants and detected for filaria using PCR, targeting the region of the internal transcribed spacer 2 (ITS2), the cytochrome c oxidase subunit 1 (cox1), and the RNA polymerase II large subunit (rbp1). In total, 4.33% (9 out of 208) of the sampled elephants had Loxodontofilaria spp. DNA with 100% query coverage. In addition, the obtained cox1 and rbp1 sequences matched with Loxodontofilaria sp., Onchocerca sp., and Dirofilaria sp. There were no identified risk factors (sex, age, location, and packed cell volume) related to Loxodontofilaria infection in elephants. The analyses of the phylogeny of ITS2 sequences demonstrated that the Loxodotofilaria-positive sequences were closely related to Onchocerca dewittei japonica and Onchocerca dewittei dewittei with 100% query coverage. Notably, the concatenated phylogenetic trees of ITS2 and the cox1 and rbp1 genes were closely similar to Loxodontofilaria sp. To describe in detail the genomic DNA of Loxodontofilaria spp., other genes should be additionally studied using a more discriminatory technique, such as DNA barcoding or whole genome sequencing.

Genome-wide SNP analysis provides insights into the XX/XY sex-determination system in silver barb (Barbonymus gonionotus).

Silver barb (Barbonymus gonionotus) is among the most economically important freshwater fish species in Thailand. It ranks fourth in economic value and third in production weight for fisheries and culture in Thailand. An XX/XY sex-determination system based on gynogenesis was previously reported for this fish. In this study, the molecular basis underlying the sex-determination system was further investigated. Genome-wide single-nucleotide polymorphism data were generated for 32 captive-bred silver barb individuals, previously scored by phenotypic sex, to identify sex-linked regions associated with sex determination. Sixty-three male-linked loci, indicating putative XY chromosomes, were identified. Male-specific loci were not observed, which indicates that the putative Y chromosome is young and the sex determination region is cryptic. A homology search revealed that most male-linked loci were homologous to the Mariner/Tc1 and Gypsy transposable elements and are probably the remnants of an initial accumulation of repeats on the Y chromosome from the early stages of sex chromosome differentiation. This research provides convincing insights into the mechanism of sex determination and reveals the potential sex determination regions in silver barb. The study provides the basic data necessary for increasing the commercial value of silver barbs through genetic improvements.

The Use of "Tail-Pedometers" to Evaluate the Impact of Dipterans in Feeder Cattle.

Hematophagous flies are a pest for livestock; their direct impact reduces productivity, and they are vectors of parasites, bacteria and viruses. Their control using insecticides is inefficient and highly polluting. The validation of new control tools requires efficacy and cost-effectiveness evaluation. The quantification of hematophagous insects' impact in livestock is a challenging prerequisite. Tail flicks counts can reliably evaluate fly-burden; however, visual records are tedious and time-consuming. In the present study, automation of tail flick counts was made through the use of pedometers attached to the tail, in two groups of feeder cattle. Group A was kept in a pen under the protection of a mosquito net, and Group B was kept in an open-air pen. The fly density of Group B was evaluated using fly traps. The apparent density per trap ranged from 130 to 1700 in the study. The mean pedometer records per 24 h ranged from 957+/-58 bits in Group A to 11,138+/-705 bits in Group B. The night/day records observed in Group A (200/800 bits) were drastically increased in Group B (1000-4000/4000-14,000 bits) and variable along seasons. A very high correlation was observed between fly density and visual records or pedometer records (PR). Two-hour PRs proved to be a reliable predictive tool for fly density. Moreover, the pedometers revealed an unsuspected but significant nuisance of mosquitoes, which should be thoroughly investigated.

Isolation and in vitro cultivation of Trypanosoma evansi Thai strains.

Trypanosoma evansi is a flagellate protozoan parasite responsible for "surra". To generate T. evansi antigens for serodiagnosis, parasites are generally propagated in laboratory animals before isolation. The alternation of animal models using axenic cultivation systems to produce trypomastigotes of various Trypanosoma species is currently available but has never been applied in Thailand. The isolation protocol for separation of live T. evansi trypomastigotes from animal blood components before in vitro cultivation has not been clearly documented. This study focused on validation of trypomastigote isolation method, in vitro cultivation of T. evansi Thai strains, and its virulence ability in vivo. In this study, two strains of T. evansi collected from Thailand were used. Trypanosoma evansi trypomastigotes were propagated in mice, and three different isolation methods, including: low-speed centrifugation, high-speed centrifugation, and ion exchange chromatography using diethylaminoethyl (DEAE) cellulose (or DE52), were compared. Four solutions of in vitro cultivation media, two different in vitro cultivation containers, and different trypomastigote densities for initiation of in vitro culture were compared. Virulence test using in vitro-adapted parasite for 100 days was conducted in vivo. The results showed that the DE52 isolation method was suitable for separation of live T. evansi trypomastigotes from animal blood components before conducting in vitro cultivation. Trypanosoma evansi Thai strains were successfully cultivated and multiplied in HMI-9 Solution I using 25 cm2 flasks and 12-well plates. The parasite was growing slowly at the initiation of in vitro culture for 15-16 days, and then rapidly increased to 10, 20, 50, 100, and 200 folds, approximately. The doubling times were varied from 11.95 ± 8 h to 41.18 ± 4.29 h in vitro. The maximum densities have reached from 0.14 × 106 to 4.63 × 106 trypomastigotes/ml. Virulence test showed that the in vitro-cultivated T. evansi was virulent in mice. In conclusion, T. evansi Thai strains were successfully isolated and cultivated in vitro for the first time. The isolation and in vitro cultivation protocols were clearly provided. The benefit of using the in vitro cultivation system helps in the production of T. evansi antigen, and replacing the use of experimental animals. It is also useful for the development of diagnostic tests in the future.

Development and evaluation of indirect enzyme-linked immunosorbent assay using recombinant dense granule antigen 7 protein for the detection of Toxoplasma gondii infection in cats in Thailand.

Background and Aim: Toxoplasma gondii is recognized as a zoonosis causing toxoplasmosis in animals globally. Cat is a definitive host of T. gondii and sheds oocyst through feces, which can infect human beings and animals through contaminated food ingestion. A precise diagnostic test is essential to prevent T. gondii infection in both humans and animals. This study aimed to develop and evaluate the pETite-dense granule antigen 7(GRA7)-based indirect enzyme-linked immunosorbent assay (ELISA) to detect T. gondii infection in cats. Materials and Methods: T. gondii-GRA7 was cloned and expressed in the Expresso®small ubiquitin-related modifier (SUMO) T7 Cloning and Expression System. The recombinant pETite-GRA7 was purified using HisTrap affinity chromatography and confirmed using Western blot analysis. The recombinant protein was used to develop and evaluate the indirect ELISA for T. gondii infection detection. In total, 200 cat sera were tested using pETite-GRA7-based indirect ELISA and indirect fluorescent antibody test (IFAT). The statistical analysis based on Kappa value, sensitivity, specificity, positive predictive value, negative predictive value, χ 2 test, and receiver operating characteristic (ROC) curve was used to evaluate the performance of the test. Results: A 606 bp GRA7 polymerase chain reaction (PCR) product was obtained from T. gondii RH strain genomic DNA. The gene was cloned into the pETite™ vector and transformed to HI-Control Escherichia coli BL21 (DE3) for protein expression. Approximately 35 kDa of recombinant pETite-GRA7 was observed and Western blot analysis showed positive bands against anti-6-His antibody and positive-T. gondii cat serum. A sample of 0.5 µg/mL of pETite-GRA7 was subjected to indirect ELISA to detect T. gondii infection in the cat sera. The results showed sensitivity and specificity of pETite-GRA7-based indirect ELISA at 72% and 96%, respectively. An acceptable diagnostic performance was characterized by high concordant results (94%) and substantial agreement (Kappa value=0.65) with IFAT. The seroprevalence levels of ELISA and IFAT were 10% and 9%, respectively, and were not significantly (p>0.05) different. The expected performance of ELISA at different cutoff points using the ROC curve analysis revealed 89% sensitivity and 92% specificity at the cutoff value of 0.146, with a high overall assay accuracy (area under the curve=0.94). Conclusion: In this study, the pETite™ vector, N-terminal 6xHis SUMO fusion tag, was used to improve the solubility and expression level of GRA7. The recombinant pETite-GRA7 showed enhanced protein solubility and purification without special condition requirements. This pETite-GRA7-based indirect ELISA showed high concordant results and substantial agreement with IFAT. ELISA revealed an acceptable sensitivity and specificity. These initial data obtained from cats' sera demonstrated that pETite-GRA7-based indirect ELISA could be a useful method for local serological diagnosis of T. gondii infection in cats in Thailand.

Investigation of Trypanosoma evansi infection in bullfighting cattle in Southern Thailand.

BACKGROUND AND AIM: Trypanosoma evansi infection has been reported in Thai livestock such as beef and dairy cattle. However, there is little information on T. evansi infection in bullfighting cattle in Southern Thailand. The aim of this study was to investigate the infection of T. evansi in bullfighting cattle presented for health checks at the Animal Hospital, Faculty of Veterinary Science, Prince of Songkla University, Thailand. MATERIALS AND METHODS: Blood and serum samples were collected from 177 bullfighting cattle from April 2016 to February 2017 after bullfighting matches. Animal inspected showed signs of fever, weight loss, or exercise intolerance. Investigation of T. evansi infection was tested using polymerase chain reaction (PCR) with TBR primers and using indirect enzyme-linked immunosorbent assay with T. evansi crude antigen. RESULTS: The seroprevalence of T. evansi in bullfighting cattle was 22.60% (40/177). The PCR results detected no parasite DNA in this study. However, bullfighting cattle may serve as T. evansi reservoirs. CONCLUSION: Health checking procedures for T. evansi should be promoted for bullfighting events so that infected animals can be quarantined in the preparatory stages of such events.

The Indirect ELISA Trypanosoma evansi in Equids: Optimisation and Application to a Serological Survey including Racing Horses, in Thailand.

Surra, caused by Trypanosoma evansi, is a widely distributed animal trypanosomosis; it affects both domestic and wild mammals with high economic impact. Clinical picture is moderate in bovines but severe in equids. Surra is also an important constraint for international animal trade and movements. Despite its impact, surra remains poorly diagnosed because of low sensitivity tests. To improve epidemiological knowledge of the disease and to secure international movement, efficient diagnosis tools are required. Here, we optimized and applied to equids the OIE-recommended indirect ELISA T. evansi that was validated in other species. Based on 96 positive and 1,382 negative horse reference samples from Thailand, a TG-ROC analysis was conducted to define the cutoff value. ELISA's sensitivity and specificity were estimated at 97.5% and 100%, respectively, qualifying the test to provide a reliable immune status of equids. The test was then applied on 1,961 horse samples from 18 Thai Provinces; the only scarce positives suggested that horses do not constitute a reservoir of T. evansi in Thailand. All samples from racing horses were negative. Conversely, two outbreaks of surra reported to our laboratory, originating from a bovine reservoir, exhibited high morbidity and lethality rates in horses. Finally, posttreatment follow-ups of infected animals allowed us to provide outbreak management guidelines.

Adaptation and evaluation of an ELISA for Trypanosoma evansi infection (surra) in elephants and its application to a serological survey in Thailand.

Trypanosoma evansi, the causative agent of surra, is widespread in domestic livestock and wildlife in South East Asia. Surra can affect cattle, buffaloes, horses and also Asian elephants (Elephas maximus). Despite the 'threatened to extinction' CITES status of elephant, surra's impact has not been thoroughly assessed yet in this species. This work offers to adapt an antibody enzyme-linked immunosorbent assay (ELISA) protocol, to detect Trypanosoma evansi antibodies in elephant serum. The test was validated with 365 negative-reference samples, which allowed the determination of a 16% positive threshold. The test was applied to a serological survey including 375 individuals. The estimated global seroprevalence was 2·1% (95% CI 1·1-4·2%). Therefore, surra does not appear to be endemic in Thai domestic elephants, but occasional outbreaks were reported to our laboratory during the survey period. These outbreaks seemed to be linked to close proximity to cattle or buffaloes, and led to severe clinical signs in elephants. Frequent relapses were observed after treatment with diminazene aceturate, the only trypanocide drug currently available in Thailand. Therefore, care should be taken to keep elephants away from bovine reservoirs, and to monitor the disease in this endangered species. ELISA proved to be reliable for screening purposes as well as for post-treatment monitoring.

Zoonotic trypanosomes in South East Asia: Attempts to control Trypanosoma lewisi using veterinary drugs.

A growing number of atypical human infections due to the livestock parasite Trypanosoma evansi, or to the rat parasite Trypanosoma lewisi, are reported in humans in Asia. In some cases, clinical evolutions request treatments, however, so far, there were very few attempts to control T. lewisi using trypanocidal drugs. In a study published elsewhere, the efficacy of human trypanocides is evaluated in laboratory rats, and it concludes that none of them is able to cure rats experimentally infected with T. lewisi. Control of T. lewisi in rat would be a step for identification of drugs against this parasite. In the present study, 4 veterinary drugs: diminazene aceturate, isometamidium chloride, melarsomine hydrochloride and quinapyramine sulfate and chloride, were evaluated at low and high doses, in intra-muscular injections to normal rats experimentally infected with a stock of T. lewisi from Thailand. None of these treatments being efficient, a trial was also made using melarsomine hydrochloride in T. evansi infected rats and in mixed T. lewisi and T. evansi infected rats, in order to demonstrate the efficacy of the drugs under the present protocol. T. evansi was cleared from the rat's blood the day after the treatment, while, T. lewisi remained unaffected until the end of the experiment. These observations clearly demonstrated the efficacy of melarsomine hydrochloride against T. evansi and its inefficacy against T. lewisi. In conclusion none of the veterinary drugs was efficient against this stock of T. lewisi. Other protocols using higher doses or other drugs and T. lewisi stocks should be investigated in further studies. The control of T. lewisi infection in Wistar rats, using veterinary trypanocidal drugs, remains so far unsuccessful.

A Clinical and Epidemiological Investigation of the First Reported Human Infection With the Zoonotic Parasite Trypanosoma evansi in Southeast Asia.

BACKGROUND: Trypanosomais a genus of unicellular parasitic flagellate protozoa.Trypanosoma bruceispecies and Trypanosoma cruziare the major agents of human trypanosomiasis; other Trypanosomaspecies can cause human disease, but are rare. In March 2015, a 38-year-old woman presented to a healthcare facility in southern Vietnam with fever, headache, and arthralgia. Microscopic examination of blood revealed infection with Trypanosoma METHODS: Microscopic observation, polymerase chain reaction (PCR) amplification of blood samples, and serological testing were performed to identify the infecting species. The patient's blood was screened for the trypanocidal protein apolipoprotein L1 (APOL1), and a field investigation was performed to identify the zoonotic source. RESULTS: PCR amplification and serological testing identified the infecting species as Trypanosoma evansi.Despite relapsing 6 weeks after completing amphotericin B therapy, the patient made a complete recovery after 5 weeks of suramin. The patient was found to have 2 wild-type APOL1 alleles and a normal serum APOL1 concentration. After responsive animal sampling in the presumed location of exposure, cattle and/or buffalo were determined to be the most likely source of the infection, with 14 of 30 (47%) animal blood samples testing PCR positive forT. evansi. CONCLUSIONS: We report the first laboratory-confirmed case ofT. evansiin a previously healthy individual without APOL1 deficiency, potentially contracted via a wound while butchering raw beef, and successfully treated with suramin. A linked epidemiological investigation revealed widespread and previously unidentified burden ofT. evansiin local cattle, highlighting the need for surveillance of this infection in animals and the possibility of further human cases.