Observing How Glutathione and S-Hexyl Glutathione Bind to Glutathione S-Transferase from Rhipicephalus (Boophilus) microplus.

Rhipicephalus (Boophilus) microplus is one of the most widespread ticks causing a massive loss to livestock production. The long-term use of acaracides rapidly develops acaracide resistance. In R. microplus, enhancing the metabolic activity of glutathione S-transferase (RmGST) is one of the mechanisms underlying acaracide resistance. RmGST catalyzes the conjugation of glutathione (GSH) to insecticides causing an easy-to-excrete conjugate. The active RmGST dimer contains two active sites (hydrophobic co-substrate binding site (H-site) and GSH binding site (G-site)) in each monomer. To preserve the insecticide efficacy, s-hexyl glutathione (GTX), a GST inhibitor, has been used as a synergist. To date, no molecular information on the RmGST-GSH/GTX complex is available. The insight is important for developing a novel RmGST inhibitor. Therefore, in this work, molecular dynamics simulations (MD) were performed to explore the binding of GTX and GSH to RmGST. GSH binds tighter and sits rigidly inside the G-site, while flexible GTX occupies both active sites. In GSH, the backbone mainly interacts with W8, R43, W46, K50, N59, L60, Q72, and S73, while its thiol group directs to Y7. In contrast, the aliphatic hexyl of GTX protrudes into the H-site and allows a flexible peptide core to form various interactions. Such high GTX flexibility and the protrusion of its hexyl moiety to the H-site suggest the dual role of GTX in preventing the conjugation reaction and the binding of acaracide. This insight can provide a better understanding of an important insecticide-resistance mechanism, which may in turn facilitate the development of novel approaches to tick control.

Distribution of Japanese Encephalitis Virus, Japan and Southeast Asia, 2016-2018.

During 2016-2018, we conducted surveillance for Japanese encephalitis virus (JEV) in mosquitoes and pigs in Japan, Thailand, the Philippines, and Indonesia. Phylogenetic analyses demonstrated that our isolates (genotypes Ia, Ib, III, IV) were related to JEV isolates obtained from the same regions many years ago. Indigenous JEV strains persist in Asia.

Changing landscapes of Southeast Asia and rodent-borne diseases: decreased diversity but increased transmission risks.

The reduction in biodiversity from land use change due to urbanization and agricultural intensification appears to be linked to major epidemiological changes in many human diseases. Increasing disease risks and the emergence of novel pathogens result from increased contact among wildlife, domesticated animals, and humans. We investigated the relationship between human alteration of the environment and the occurrence of generalist and synanthropic rodent species in relation to the diversity and prevalence of rodent-borne pathogens in Southeast Asia, a hotspot of threatened and endangered species, and a foci of emerging infectious diseases. We used data from an extensive pathogen survey of rodents from seven sites in mainland Southeast Asia in conjunction with past and present land cover analyses. At low spatial resolutions, we found that rodent-borne pathogen richness is negatively associated with increasing urbanization, characterized by increased habitat fragmentation, agriculture cover and deforestation. However, at a finer spatial resolution, we found that some major pathogens are favored by environmental characteristics associated with human alteration including irrigation, habitat fragmentation, and increased agricultural land cover. In addition, synanthropic rodents, many of which are important pathogen reservoirs, were associated with fragmented and human-dominated landscapes, which may ultimately enhance the opportunities for zoonotic transmission and human infection by some pathogens.

Getah virus epizootic among wild boars in Japan around 2012.

In 2014, an outbreak of Getah virus (GETV) infection occurred in Japan in a horse population that was inoculated with a vaccine against GETV. In this study, we investigated the seroprevalence of GETV infection among wild boars in Japan. Interestingly, the highest rate of anti-GETV-positive wild boars was observed in 2013, which gradually decreased during 2014-2016. The results suggested that GETV spread among wild boars around 2012, resulting in the 2014 outbreak.

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.

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.

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.

Molecular detection of Cryptosporidium spp. infections in water buffaloes from northeast Thailand.

The objectives of this study were to determine the individual and herd-level prevalence and genotype of Cryptosporidium and to identify putative risk factors associated with Cryptosporidium spp. infections in water buffaloes in northeast Thailand. Fecal samples from 600 water buffaloes of 287 farms in six provinces were collected and tested using DMSO-modified acid-fast staining and polymerase chain reaction. The overall prevalence of Cryptosporidium infections in buffaloes was 5.7 and 8.7% among individual animals and herds, respectively. The provinces with highest infected Cryptosporidium were located in the Sakon Nakhon Basin in the northern part of the region. In addition, higher herd prevalence was observed among farms with more than five buffaloes (30%) than those with five or less animals (16.2%). Thirty (88.2%) of the 34 Cryptosporidium-positive samples were Cryptosporidium parvum and four (11.8%) were Cryptosporidium ryanae.

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.

Elucidating structure and dynamics of glutathione S-transferase from Rhipicephalus (Boophilus) microplus.

Rhipicephalus (Boophilus) microplus is tick parasite that affects the cattle industry worldwide. In R. (B.) microplus, acaricide resistance develops rapidly against many commercial acaricides. One of main resistance strategies is to enhance the metabolic detoxification mediated by R. (B.) microplus glutathione-S-transferase (RmGST). RmGST detoxifies acaricides by catalyzing the conjugation of glutathione to acaricides. Although structural and dynamic details of RmGST are expected to elucidate the biologic activity of this molecule, these data have not been available to date. Thus, Molecular Dynamics simulations were employed to study ligand-free RmGST at an atomic level. Like other m-class GSTs, the flexible m loop (m1) of RmGST was observed. M1 seems to shield the active sites from the bulk. A RmGST dimer is stabilized by the lock-and-key motif (F57 as "key") and hydrogen bonds of R82-E91 and R82-D98 at the dimer interface. Without substrates, conserved catalytic Y116 and N209 can interact with V112, G210 (for Y116) and F215 (for N209). Overall, most residues involving in RmGST function and stability are similar to other m-class GSTs. This implies similar structural stability and catalytic activity of RmGST to other GSTs. An insight obtained here will be useful for management of acaricide resistance and tick control.Communicated by Ramaswamy H. Sarma.

Characterization and Homology Modeling of Catalytically Active Recombinant PhaCAp Protein from Arthrospira platensis.

Polyhydroxybutyrate (PHB) is a biocompatible and biodegradable polymer that has the potential to replace fossil-derived polymers. The enzymes involved in the biosynthesis of PHB are ß-ketothiolase (PhaA), acetoacetyl-CoA reductase (PhaB), and PHA synthase (PhaC). PhaC in Arthrospira platensis is the key enzyme for PHB production. In this study, the recombinant E. cloni®10G cells harboring A. platensis phaC (rPhaCAp) was constructed. The overexpressed and purified rPhaCAp with a predicted molecular mass of 69 kDa exhibited Vmax, Km, and kcat values of 24.5 ± 2 µmol/min/mg, 31.3 ± 2 µM and 412.7 ± 2 1/s, respectively. The catalytically active rPhaCAp was a homodimer. The three-dimensional structural model for the asymmetric PhaCAp homodimer was constructed based on Chromobacterium sp. USM2 PhaC (PhaCCs). The obtained model of PhaCAp revealed that the overall fold of one monomer was in the closed, catalytically inactive conformation whereas the other monomer was in the catalytically active, open conformation. In the active conformation, the catalytic triad residues (Cys151-Asp310-His339) were involved in the binding of substrate 3HB-CoA and the CAP domain of PhaCAp involved in the dimerization.

High Seroprevalence of Severe Fever with Thrombocytopenia Syndrome Virus Infection among the Dog Population in Thailand.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne zoonotic disease caused by the SFTS virus (SFTSV). In Thailand, three human cases of SFTS were reported in 2019 and 2020, but there was no report of SFTSV infection in animals. Our study revealed that at least 16.6% of dogs in Thailand were seropositive for SFTSV infection, and the SFTSV-positive dogs were found in several districts in Thailand. Additionally, more than 70% of the serum samples collected at one shelter possessed virus-neutralization antibodies against SFTSV and the near-complete genome sequences of the SFTSV were determined from one dog in the shelter. The dog SFTSV was genetically close to those from Thailand and Chinese patients and belonged to genotype J3. These results indicated that SFTSV has already spread among animals in Thailand.

Prevalence and genetic diversity of Bartonella spp. in small mammals from Southeastern Asia.

Among 1,341 blood samples from rodents that were trapped in Southeast Asia between 2008 and 2010, we found a prevalence of Bartonella infection ranging from 9.6 to 11.9%. Bartonella species identified (143 isolates) included B. elizabethae, B. coopersplainsensis, B. phoceensis, B. queenslandensis, B. rattimassiliensis, B. tribocorum, and three new putative Bartonella species.

Molecular detection and identification of Babesia bovis and Babesia bigemina in cattle in northern Thailand.

Although Babesia bovis and Babesia bigemina infections cause economic losses in the cattle industry in northern Thailand, there is inadequate information on Babesia isolates present in the area. Therefore, to determine the prevalence and genetic relationship between Babesia isolates, we screened 200 blood samples of cattle from Chiang Rai, Chiang Mai, and Lumpang provinces of northern Thailand. A nested polymerase chain reaction using primers targeting B. bovis spherical body protein 2 (BboSBP2) and B. bigemina rhoptry-associated protein 1a (BbiRAP-1a) genes revealed a prevalence of 12 and 21 % for B. bovis and B. bigemina, respectively, while that of mixed infections was 6.5 % samples. The prevalences of B. bovis in Chiang Rai, Chiang Mai, and Lumpang were 9.5, 3.7, and 25.5 %, respectively. For B. bigemina, the prevalences were 15.8, 12.9, and 39.2 % in Chiang Rai, Chiang Mai, and Lumpang, respectively. Mixed infections with B. bovis and B. bigemina were 6.3 % in Chiang Rai, 1.9 % in Chiang Mai, and 13.7 % in Lumpang. The identical sequences of either BboSBP2 gene or BbiRAP-1a gene were shared among the Babesia isolates in the three provinces of northern Thailand. Further analysis using the internal transcribed spacer gene revealed at least four genotypes for B. bovis and five genotypes for B. bigemina in northern Thailand, while the sequences present great genetic diversities in the different isolates. Overall, we have demonstrated a high prevalence and polymorphism of Babesia parasites in northern Thailand calling for the need to design effective control programs for bovine babesiosis.

Risk factors of Neospora caninum infection in dogs and cats in dairy farms in Western Thailand.

The objectives of this study were to investigate the risk factors of Neospora caninum infection in dogs and cats found in dairy herds in western Thailand. A case-control study was conducted in dairy herds in three western provinces including Nakhon Pathom, Ratchaburi and Kanchanaburi. Blood samples of pets from 14 positive dairy herds and 26 herds from negative neighbouring farms which were randomly selected, in total blood samples from dogs and cats from 40 herds were collected and examined for antibodies against N. caninum infections using competitive ELISA and indirect fluorescent antibody test (IFAT). No seropositive cats were found. Of the 38 positive dogs, four (10.5%) were seropositive to N. caninum, which was higher than the proportion of seropositive in the negative herd population (one of 76, 1.3%). The higher proportion of seropositive farm dogs as compared with neighbouring dogs tended to be significant (P = 0.055).

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.

Plasmidome in mcr-1 harboring carbapenem-resistant enterobacterales isolates from human in Thailand.

The emergence of the mobile colistin-resistance genes mcr-1 has attracted significant attention worldwide. This study aimed to investigate the genetic features of mcr-1-carrying plasmid among carbapenem-resistant Enterobacterales (CRE) isolates and the potential genetic basis governing transmission. Seventeen mcr-harboring isolates were analyzed based on whole genome sequencing using short-read and long-read platforms. All the mcr-1-carrying isolates could be conjugatively transferred into a recipient Escherichia coli UB1637. Among these 17 isolates, mcr-1 was located on diverse plasmid Inc types, consisting of IncX4 (11/17; 64.7%), IncI2 (4/17; 23.53%), and IncHI/IncN (2/17; 11.76%). Each of these exhibited remarkable similarity in the backbone set that is responsible for plasmid replication, maintenance, and transfer, with differences being in the upstream and downstream regions containing mcr-1. The IncHI/IncN type also carried other resistance genes (blaTEM-1B or blaTEM-135). The mcr-1-harboring IncX4 plasmids were carried in E. coli ST410 (7/11; 63.6%) and ST10 (1/11; 9.1%) and Klebsiella pneumoniae ST15 (1/11; 9.1%), ST336 (1/11; 9.1%), and ST340 (1/11; 9.1%). The IncI2-type plasmid was harbored in E. coli ST3052 (1/4; 25%) and ST1287 (1/4; 25%) and in K. pneumoniae ST336 (2/4; 50%), whereas IncHI/IncN were carried in E. coli ST6721 (1/2; 50%) and new ST (1/2; 50%). The diverse promiscuous plasmids may facilitate the spread of mcr-1 among commensal E. coli or K. pneumoniae strains in patients. These results can provide information for a surveillance system and infection control for dynamic tracing.

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.

A review on the diagnosis of animal trypanosomoses.

This review focuses on the most reliable and up-to-date methods for diagnosing trypanosomoses, a group of diseases of wild and domestic mammals, caused by trypanosomes, parasitic zooflagellate protozoans mainly transmitted by insects. In Africa, the Americas and Asia, these diseases, which in some cases affect humans, result in significant illness in animals and cause major economic losses in livestock. A number of pathogens are described in this review, including several Salivarian trypanosomes, such as Trypanosoma brucei sspp. (among which are the agents of sleeping sickness, the human African trypanosomiasis [HAT]), Trypanosoma congolense and Trypanosoma vivax (causing "Nagana" or animal African trypanosomosis [AAT]), Trypanosoma evansi ("Surra") and Trypanosoma equiperdum ("Dourine"), and Trypanosoma cruzi, a Stercorarian trypanosome, etiological agent of the American trypanosomiasis (Chagas disease). Diagnostic methods for detecting zoonotic trypanosomes causing Chagas disease and HAT in animals, as well as a diagnostic method for detecting animal trypanosomes in humans (the so-called "atypical human infections by animal trypanosomes" [a-HT]), including T. evansi and Trypanosoma lewisi (a rat parasite), are also reviewed. Our goal is to present an integrated view of the various diagnostic methods and techniques, including those for: (i) parasite detection; (ii) DNA detection; and (iii) antibody detection. The discussion covers various other factors that need to be considered, such as the sensitivity and specificity of the various diagnostic methods, critical cross-reactions that may be expected among Trypanosomatidae, additional complementary information, such as clinical observations and epizootiological context, scale of study and logistic and cost constraints. The suitability of examining multiple specimens and samples using several techniques is discussed, as well as risks to technicians, in the context of specific geographical regions and settings. This overview also addresses the challenge of diagnosing mixed infections with different Trypanosoma species and/or kinetoplastid parasites. Improving and strengthening procedures for diagnosing animal trypanosomoses throughout the world will result in a better control of infections and will significantly impact on "One Health," by advancing and preserving animal, human and environmental health.

Diagnosis of animal trypanosomoses: proper use of current tools and future prospects.

Reliable diagnostic tools are needed to choose the appropriate treatment and proper control measures for animal trypanosomoses, some of which are pathogenic. Trypanosoma cruzi, for example, is responsible for Chagas disease in Latin America. Similarly, pathogenic animal trypanosomoses of African origin (ATAO), including a variety of Trypanosoma species and subspecies, are currently found in Africa, Latin America and Asia. ATAO limit global livestock productivity and impact food security and the welfare of domestic animals. This review focusses on implementing previously reviewed diagnostic methods, in a complex epizootiological scenario, by critically assessing diagnostic results at the individual or herd level. In most cases, a single diagnostic method applied at a given time does not unequivocally identify the various parasitological and disease statuses of a host. These include "non-infected", "asymptomatic carrier", "sick infected", "cured/not cured" and/or "multi-infected". The diversity of hosts affected by these animal trypanosomoses and their vectors (or other routes of transmission) is such that integrative, diachronic approaches are needed that combine: (i) parasite detection, (ii) DNA, RNA or antigen detection and (iii) antibody detection, along with epizootiological information. The specificity of antibody detection tests is restricted to the genus or subgenus due to cross-reactivity with other Trypanosoma spp. and Trypanosomatidae, but sensitivity is high. The DNA-based methods implemented over the last three decades have yielded higher specificity and sensitivity for active infection detection in hosts and vectors. However, no single diagnostic method can detect all active infections and/or trypanosome species or subspecies. The proposed integrative approach will improve the prevention, surveillance and monitoring of animal trypanosomoses with the available diagnostic tools. However, further developments are required to address specific gaps in diagnostic methods and the sustainable control or elimination of these diseases.