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.

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.

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.