Molecular prevalence and associated risk factors of Cryptosporidium spp. infection in dairy cattle in Khon Kaen, Thailand.

Background and Aim: Cryptosporidium spp. are important parasites in the small intestines of humans and animals, particularly cattle. The aim of this study was to estimate the molecular prevalence and associated risk factors of Cryptosporidium infection in dairy cattle in five districts of Khon Kaen province, Thailand, and to identify Cryptosporidium spp. Materials and Methods: From July 2020 to October 2021, 296 fecal samples were collected from three groups of dairy cattle: Calves aged <3 months, calves aged 3 months-1 year, and calves aged >1 year. Cryptosporidium spp. were detected by polymerase chain reaction (PCR) amplifying the 18s RNA gene. Both genus-specific and species-specific primers were used to identify Cryptosporidium confirmed by DNA sequencing. Age, house floor type, and water trough type were evaluated as risk factors. We analyzed all associated risk factor information using the logistic regression test in the Statistical Package for the Social Sciences. Results: PCR results showed that 40 (13.51%) out of 296 samples were positive for Cryptosporidium spp., including Cryptosporidium bovis (57.50%) and Cryptosporidium ryanae (2.50%). There was a significant association between Cryptosporidium incidence, cattle age, and house floor type (p < 0.05). National Center for Biotechnology Information Basic Local Alignment Search Tool displayed 99.48%-100% nucleotide similarity of each Cryptosporidium spp. isolate with references recorded on GenBank. Conclusion: C. bovis and C. ryanae are commonly found in dairy cattle, especially calves, in Khon Kaen, Thailand, and the incidence was associated with age and house floor type. A molecular technique may be influential for species identification. The results of the present study would provide useful information for veterinarians and animal owners to understand better Cryptosporidium spp. and how to manage farms properly.

First report on the molecular detection and genetic diversity of Anaplasma marginale in healthy dairy cattle in Khon Kaen province, Thailand.

Background and Aim: Bovine anaplasmosis (BA) is one of the most important diseases of ruminants worldwide, causing significant economic losses in the livestock industry due to the high morbidity and mortality in susceptible cattle herds. Anaplasma marginale is the main causative agent of BA occurring worldwide in tropical and subtropical regions. This study aimed to investigate the first molecular detection and genetic diversity of A. marginale in dairy cattle in Khon Kaen Province, Thailand. Materials and Methods: Blood samples were collected from 385 lactating cows from 40 dairy farms in five districts of Khon Kaen, regardless of age and health status. To detect A. marginale, all DNA preparations were used for molecular diagnosis using a single polymerase chain reaction with the msp4 gene target. A phylogenetic tree was constructed from the msp4 gene sequences using molecular genetic characterization. Genetic diversity was calculated as haplotype diversity, haplotype number, number of nucleotide differences, nucleotide diversity, and average number of nucleotide differences. Results: The overall prevalence of A. marginale was 12.72% (49/385). The highest prevalence (17.19%) was found in Ubolratana district, followed by Muang, Kranuan, Khao Suan Kwang, and Nam Phong districts (14.94%, 14.74%, 13.79%, and 3.70%, respectively). Phylogenetic analysis showed that A. marginale was closely related to isolates from Australia (98.96%), China (99.68%), Spain (99.74%), and the USA (99.63%). Conclusion: The molecular prevalence of BA in dairy cattle is the first to be observed in this area, and the genetic variability with separated clusters shown in the msp4 gene of A. marginale revealed species variation in dairy cattle. This significant genetic diversity contributes to the understanding of the diversity of A. marginale and will be important for the control and prevention of A. marginale in dairy cattle.

First report on the molecular prevalence and associated risk factors of Eimeria spp. in dairy cattle in Khon Kaen, Thailand.

Background and Aim: Bovine coccidiosis, caused by the protozoa Eimeria, is an important parasitic cattle disease that affects animal health and has economic impact worldwide. This study was conducted to report the first molecular prevalence and genetic diversity of Eimeria spp. in dairy cattle in Khon Kaen province, Thailand, and to identify the risk factors associated with Eimeria spp. infection. Materials and Methods: From July 2020 to October 2021, 296 fecal samples were collected from dairy cattle divided into three age groups, including <3-month-old calves, 3-month-old to 1-year-old calves, and >1-year-old cattle. Eimeria spp. were identified by multiplex polymerase chain reaction (PCR) amplifying 18S RNA gene and confirmed by DNA sequencing. Information regarding all associated risk factors was collected using questionnaires and analyzed using logistic regression tests in the Statistical Package for the Social Sciences program. Results: Polymerase chain reaction results showed that 104 (35.13%) of 296 samples were positive for Eimeria spp. The <3-month-old calves (46.51%) had the highest infection rate. Moreover, multiplex PCR identified five species of Eimeria, namely, Eimeria bovis (32.69%), Eimeria zuernii (18.26%), Eimeria alabamensis (5.76%), Eimeria ellipsoidalis (3.84%), and Eimeria cylindrica (2.88%). An association was observed between risk factors and Eimeria spp. incidence (p < 0.05). DNA sequencing revealed the similarity of each Eimeria spp. with 91%-100% nucleotide identity. Phylogenetic tree analysis demonstrated the close relationships of clusters of E. bovis and E. zuernii, E. ellipsoidalis, and E. cylindrica and another cluster of E. alabamensis. Conclusion: The results confirm that Eimeria spp. are commonly found in dairy cattle, especially calves. The molecular test could be powerful for species identification. This study also provides epidemiological information for developing future strategies to control bovine coccidiosis.

The long-term gut bacterial signature of a wild primate is associated with a timing effect of pre- and postnatal maternal glucocorticoid levels.

BACKGROUND: During development, elevated levels of maternal glucocorticoids (GCs) can have detrimental effects on offspring morphology, cognition, and behavior as well as physiology and metabolism. Depending on the timing of exposure, such effects may vary in strength or even reverse in direction, may alleviate with age, or may concern more stable and long-term programming of phenotypic traits. Maternal effects on gut bacterial diversity, composition, and function, and the persistence of such effects into adulthood of long-lived model species in the natural habitats remain underexplored. RESULTS: In a cross-sectional sample of infant, juvenile, and adult Assamese macaques, the timing of exposure to elevated maternal GCs during ontogeny was associated with the gut bacterial community of the offspring. Specifically, naturally varying maternal GC levels during early but not late gestation or lactation were associated with reduced bacterial richness. The overall effect of maternal GCs during early gestation on the gut bacterial composition and function exacerbated with offspring age and was 10 times stronger than the effect associated with exposure during late prenatal or postnatal periods. Instead, variation in maternal GCs during the late prenatal or postnatal period had less pronounced or less stable statistical effects and therefore a weaker effect on the entire bacterial community composition, particularly in adult individuals. Finally, higher early prenatal GCs were associated with an increase in the relative abundance of several potential pro-inflammatory bacteria and a decrease in the abundance of Bifidobacterium and other anti-inflammatory taxa, an effect that exacerbated with age. CONCLUSIONS: In primates, the gut microbiota can be shaped by developmental effects with strong timing effects on plasticity and potentially detrimental consequences for adult health. Together with results on other macaque species, this study suggests potential detrimental developmental effects similar to rapid inflammaging, suggesting that prenatal exposure to high maternal GC concentrations is a common cause underlying both phenomena. Our findings await confirmation by metagenomic functional and causal analyses and by longitudinal studies of long-lived, ecologically flexible primates in their natural habitat, including developmental effects that originate before birth. Video Abstract.

Protective efficacy of new-generation anticoccidial vaccine candidates against Eimeria infection in chickens: A meta-analysis of challenge trials.

Limitations of live oocyst anticoccidial vaccines in poultry have led to a search for the new generation of anticoccidial vaccines. Several sources may influence the protective efficacy of the new generation of anticoccidial vaccine candidates. Therefore, the objective of this study was to explore the sources influencing the protective efficacy (in terms of the lesion score and the oocyst output) of the new-generation anticoccidial vaccine candidates from the challenge trials in chickens, using meta-analysis techniques. The overall effect size was also estimated to get the overview of the protective efficacy. The study outcomes were the standardized mean difference (SMD) of the lesion score and the difference in mean (DM) of the oocyst output. Descriptive statistics of the oocyst decrease ratio (%) and anticoccidial index (ACI) were also presented. Relevant citations were retrieved from PubMed, Scopus, and Web of Science. Of 1524 retrieved citations, 63 were included for meta-analysis (60 for the lesion score and 44 for the oocyst output). Overall, the new generation of anticoccidial vaccine candidates partially protected chickens from coccidiosis because they significantly reduced the lesion score (SMD = -3.69, [95% CI: -4.08 to -3.29], P < 0.001) with high heterogeneity (I2 = 96.85%) and the oocyst output (DM = -1.48, [95% CI: -1.75 to -1.21], P < 0.001) with high heterogeneity (I2 = 99.69%). The median oocyst decrease ratio was 66.15% (a range from 4.27% to 95.93%, n = 125 subgroups). The median ACI was 164.71 (a range from 50.05 to 196.40, n = 115 subgroups). Vaccine platform and route of administration were identified as sources of heterogeneity for the lesion score and the oocyst output. However, severe publication bias threatened validity of the lesion score outcome. After accounting for other sources of variation, the anticoccidial vaccine candidates were shown to be less effective in reducing the oocyst output when the challenge dose, the length between the day of last immunization and the day of the challenge, or the length between the day of the challenge and the day of sampling, increased. In conclusion, although the new generation of anticoccidial vaccine candidates clearly showed a partial protection of chickens from coccidiosis in experimental trials, the protective efficacy was influenced by several sources, such as the vaccine platform and route of administration. Sources of high heterogeneity such as protein antigens are worth exploring when additionally relevant data are available. Therefore, additional experimental trials in chickens are required to better understand the protective efficacy of the new-generation anticoccidial vaccine candidates.

First report on molecular characteristics and risk factor analysis of Ehrlichia canis in dogs in Khon Kaen, Thailand.

Background and Aim: Ehrlichia canis is a well-known cause of both anemia and thrombocytopenia in dogs. There are insufficient epidemiological data on this blood parasite in Thailand and the association of infections with hematological abnormalities. This study aimed to analyze the molecular characteristics and to identify E. canis as well as the risk factors associated with E. canis infection in dogs in Khon Kaen, Thailand. Materials and Methods: Blood samples from 126 dogs that visited animal clinics were subjected to molecular detection using nested polymerase chain reaction for E. canis 16S rRNA gene. The risk factors and hematological profiles associated with the infection were analyzed using the logistic regression test in program SPSS version 19. Results: Forty-one dogs were infected, indicating a 32.5% molecular infection rate of E. canis. The factors significantly associated with E. canis infection include animal housing status, low packed cell volume, low red blood cell count, and low platelets (p<0.05). Ten positive samples were amplified, sequenced, and phylogenetically analyzed. Sequence and phylogenetic analysis confirmed the current ten samples as E. canis compared with reference sequences in GenBank, using the BLAST program hosted by NCBI, which showed 99.74-100% similarity. Conclusion: This study provided the first data of infection rate of E. canis using nested PCR and molecular characteristics of E. canis in randomly selected domestic dogs in Khon Kaen, Thailand.

First report on molecular prevalence and identification of Anaplasma platys in dogs in Khon Kaen, Thailand.

BACKGROUND AND AIM: Anaplasma platys is a blood parasite that infects platelets, causing thrombocytopenia. Rhipicephalus sanguineus ticks are believed to transmit A. platys. To identify A. platys, nested polymerase chain reaction (PCR) has proven to be an effective diagnostic tool. In this study, the molecular prevalence of A. platys infection in dogs was investigated for the 1st time in the Khon Kaen region of Thailand. The association between risk factors and A. platys infection was also evaluated. MATERIALS AND METHODS: A total of 130 blood samples were collected from dogs in Khon Kaen, Thailand. DNA from the samples was extracted and nested PCR was applied for molecular analysis. Platelet count and packed cell volume (PCV) levels were measured. Platelet counts were categorized into four grades: Non-thrombocytopenia (platelets >200,000 cells/µL), mild thrombocytopenia (platelets 150,000-200,000 cells/µL), moderate thrombocytopenia (platelets 100,000-150,000 cells/µL), and severe thrombocytopenia (platelets <100,000 cells/µL). Four categories for PCV levels of >37%, 30-37%, 20-29%, and <20% were defined as no anemia, mild anemia, moderate anemia, and severe anemia, respectively. DNA sequencing was analyzed using BTSeq™ (Barcode-Tagged Sequencing; CELEMICS, Seoul, South Korea) for similarity index. RESULTS: Among the 130 samples, 9 (6.9%) were positive for A. platys infection. There was an association between low platelet count and infection (p<0.05). PCV level was also associated with A. platys infection (p<0.05). DNA sequencing results of the nine positive samples showed similarity to known sequences of A. platys with 99.36-100% nucleotide identity. These results suggested low genetic diversity in A. platys infecting dogs in the Khon Kaen area. CONCLUSION: By amplifying 16S rRNA, A. platys infection was detected in the blood of Thai dogs. Further work should be performed to identify risk factors potentially associated with A. platys infection in dogs in Khon Kaen. Other related factors should also be considered, such as location and breeding, as well as the environmental characteristics of each locality. In addition, sampling a larger number of animals may reveal predictors for the positivity of A. platys in dogs in this region.

Metabarcoding of eukaryotic parasite communities describes diverse parasite assemblages spanning the primate phylogeny.

Despite their ubiquity, in most cases little is known about the impact of eukaryotic parasites on their mammalian hosts. Comparative approaches provide a powerful method to investigate the impact of parasites on host ecology and evolution, though two issues are critical for such efforts: controlling for variation in methods of identifying parasites and incorporating heterogeneity in sampling effort across host species. To address these issues, there is a need for standardized methods to catalogue eukaryotic parasite diversity across broad phylogenetic host ranges. We demonstrate the feasibility of a metabarcoding approach for describing parasite communities by analysing faecal samples from 11 nonhuman primate species representing divergent lineages of the primate phylogeny and the full range of sampling effort (i.e. from no parasites reported in the literature to the best-studied primates). We detected a number of parasite families and regardless of prior sampling effort, metabarcoding of only ten faecal samples identified parasite families previously undescribed in each host (x̅ = 8.5 new families per species). We found more overlap between parasite families detected with metabarcoding and published literature when more research effort-measured as the number of publications-had been conducted on the host species' parasites. More closely related primates and those from the same continent had more similar parasite communities, highlighting the biological relevance of sampling even a small number of hosts. Collectively, results demonstrate that metabarcoding methods are sensitive and powerful enough to standardize studies of eukaryotic parasite communities across host species, providing essential new tools for macroecological studies of parasitism.

Prevalence and species identification of Cryptosporidium spp. in the newborn dairy calves from Muang District, Khon Kaen Province, Thailand.

AIM: This study aims to determine the prevalence of Cryptosporidium spp. infection and to identify the species of Cryptosporidium spp. in newborn dairy calves between December 2016 and March 2017 in Muang District, Khon Kaen Province, Thailand. MATERIALS AND METHODS: A total of 200 fecal samples from newborn dairy calves of the ages 1 day up to 28 days were collected and the presence of Cryptosporidium oocysts was examined microscopically using the modified Kinyoun's acid-fast staining technique. Then, Cryptosporidium species were identified using nested polymerase chain reaction amplification of 18S rRNA gene and sequencing. RESULTS: The modified Kinyoun's acid-fast staining revealed the presence of Cryptosporidium oocysts in 51% (102/200). Sequence analysis of the 18S rRNA gene identified two species, namely, Cryptosporidium bovis (n=11) and Cryptosporidium ryanae (n=11) and one isolated strain could not be identified. CONCLUSION: This study indicated that newborn dairy calves aging up to 4 weeks were highly infected with Cryptosporidium spp., and the infection mostly occurred in diarrheic dairy calves. This is the first report of Cryptosporidium in dairy calves in Khon Kaen Province and the results provide baseline information for further studies and control of Cryptosporidium infection in dairy calves in the study area.

Promotion of Dental Pulp Wound Healing in New Zealand White Rabbits' Teeth by Thai Propolis Product.

This study examined and compared wound healing between Thai propolis product and calcium hydroxide paste as pulp-capping agents after partial pulpotomy in New Zealand white rabbits. Forty incisor teeth from 10 rabbits were treated. Thirty-six teeth received class V cavity preparations with partial pulpotomy and application of either propolis or calcium hydroxide paste. Similar cavity preparations were performed in 2 teeth without any capping material as a positive control, whereas 2 teeth without the cavity preparation served as a negative control. Histological evaluation showed that both groups had dentin bridge formation. Dentinal tubules in the dentin bridge were more orderly arranged in the Thai propolis group than in the calcium hydroxide group. Wound healing and the median number of hyperemic blood vessels were not statistically significant different between the 2 groups. Thai propolis product may be used as a pulp-capping agent.

Molecular detection and treatment of tick-borne pathogens in domestic dogs in Khon Kaen, northeastern Thailand.

We determined the prevalence of tick-borne pathogens in domestic dogs using microscopy and polymerase chain reaction (PCR) techniques. A total of 303 EDTA blood samples were collected from domestic dogs in Khon Kaen Province, Thailand, in May 2013. Microscopic observation of Giemsa-stained smears and molecular diagnosis using conventional PCR were performed. Infected dogs were treated with imidocarb dipropionate, a combination of imidocarb dipropionate and doxycycline, or doxycycline alone. Seventy-one (23.4%) out of 303 dogs were positive for DNA of tick-borne pathogens. Of the 303 animals, 13.2% and 1.3% were positive for a single infection with Babesia spp or Ehrlichia canis, respec- tively using microscopy; whereas 19.5% and 3.0% were positive using the PCR technique. Co-infection with Babesia spp and E. canis was observed in 0.7%, and coinfection with Hepatozoon canis and E. canis in 0.3%. Infected dogs were treated with the assigned drugs, and elimination of the pathogens was demonstrated by microscopy and PCR. The results indicated that while both microscopic and PCR diagnostic techniques were useful for tick-borne pathogen detection, PCR was more effective. Imidocarb dipropionate and doxycycline were found to be effective for treatment of babesiosis and ehrlichiosis, respectively. The present study suggests that the PCR technique has high sensitivity and specificity for Babesia and Ehrlichia diagnosis as well as for detection of Babesia spp, E. canis and H. canis DNA in EDTA blood specimens.

Molecular detection and identification of Bartonella species in rat fleas from northeastern Thailand.

The presence of Bartonella species in Xenopsylla cheopis fleas collected from Rattus spp. (R. exulans, R. norvegicus, and R. rattus) in Khon Kaen Province, Thailand was investigated. One hundred ninety-three fleas obtained from 62 rats, were screened by polymerase chain reaction using primers specific for the 16S-23S intergenic spacer region, and the presence of Bartonella DNA was confirmed by using the citrate synthase gene. Bartonella DNA was detected in 59.1% (114 of 193) of fleas examined. Sequencing demonstrated the presence of Bartonella spp. similar to B. elizabethae, B. rattimassiliensis, B. rochalimae, and B. tribocorum in the samples tested with a cutoff for sequence similarity ≥ 96% and 4 clustered together with the closest match with B. grahamii (95.5% identity). If X. cheopis proves to be a competent vector of these species, our results suggest that humans and animals residing in this area may be at risk for infection by several zoonotic Bartonella species.

Bartonella species in dogs and their ectoparasites from Khon Kaen Province, Thailand.

In order to access the prevalence of Bartonella species in dogs, whole blood and any associated ectoparasites were collected from 164 dogs with owners in 25 villages throughout Khon Kaen Province. DNA was extracted from dog blood, 92 ticks (Rhipicephalus sanguineus) and 137 fleas (Ctenocephalides spp) and screened by PCR using intergenic spacer region and citrate synthase gene primers. B. clarridgeiae DNA was detected in blood of 3 dogs, 4 C. felis and 1 C. canis; B. rochalimae DNA was found in 1 tick; and B. vinsonii subsp vinsonii DNA was found in 2 C. felis. The findings indicate that dogs residing in northeast Thailand are exposed to diverse Bartonella species that are also potential human pathogens.

Enrichment culture and molecular identification of diverse Bartonella species in stray dogs.

Using pre-enrichment culture in Bartonella alpha-Proteobacteria growth medium (BAPGM) followed by PCR amplification and DNA sequence identification that targeted a fragment of the citrate synthase gene (gltA), we provide evidence of common bartonella infections and diverse Bartonella species in the blood of stray dogs from Bangkok and Khon Kaen, Thailand. The overall prevalence of all Bartonella species was 31.3% (60/192), with 27.9% (31/111) and 35.8% (29/81) in the stray dogs from Bangkok and Khon Kaen, respectively. Phylogenetic analyzes of gltA identified eight species/genotypes of Bartonella in the blood of stray dogs, including B. vinsonii subsp. arupensis, B. elizabethae, B. grahamii, B. quintana, B. taylorii, and three novel genotypes (BK1, KK1 and KK2) possibly representing unique species with ≤ 90.2% similarities to any of the known Bartonella species B. vinsonii subsp. arupensis was the only species detected in dogs from both sites, B. quintana and BK1 were found in the dogs from Bangkok, B. elizabethae, B. taylorii, KK1 and KK2 were found in the dogs from Khon Kaen. We conclude that stray dogs in Thailand are frequently infected with Bartonella species that vary with geographic region. As some Bartonella species detected in the present study are considered pathogenic for humans, stray dogs in Thailand may serve as possible reservoirs for Bartonella causing human illnesses. Further work is needed to determine the role of those newly discovered Bartonella genotypes/species in human and veterinary medicine.

Rapid detection of Dirofilaria immitis in mosquito vectors and dogs using a real-time fluorescence resonance energy transfer PCR and melting curve analysis.

A real-time fluorescence resonance energy transfer (FRET) PCR supplemented with melting curve analysis for the rapid molecular detection of Dirofilaria immitis in mosquito vectors and dog blood samples was developed. This real-time FRET PCR was based on the fluorescence melting curve analysis of a hybrid between an amplicon generated from the D. immitis ribosomal RNA gene sequence and specific fluorophore-labeled probes. The sensitivity, specificity, accuracy, and positive and negative predictive values of this method were all 100%. Besides being highly sensitive and specific, this PCR is fast and offers a high throughput. Therefore it is a suitable and powerful tool for the diagnosis and for epidemiological surveys of canine dirofilariasis as well as for molecular xenomonitoring of D. immitis in mosquito vectors.

Immunodiagnosis of human fascioliasis using an antigen of Fasciola gigantica adult worm with the molecular mass of 27 kDa by a dot-ELISA.

Immunodominant antigens of an approximate molecular mass of 27 kDa (FG 27) were obtained from an excretory-secretory product of adult Fasciola gigantica by a simple continuous-elution method. A dot-ELISA using the FG 27 antigen was developed for the detection of specific antibodies from patients infected with F. gigantica. Control sera were obtained from patients with other parasitic infections and healthy volunteers. The accuracy, sensitivity, specificity, and positive and negative predictive values were 98.2%, 100%, 97.4%, 76.9% and 100%, respectively. This dot-ELISA is a specific, sensitive and easy to perform method for the rapid diagnosis of fascioliasis, particularly when more complex laboratory tests are unavailable.