Molecular epidemiology of Giardia duodenalis

Giardia duodenalis is a parasitic protozoan that affects the gastrointestinal tract, causing abdominal disorders of various animals and humans. To date, G. duodenalis has been genotypically divided into seven groups (assemblages), namely A to G, found in different host ranges. Whilst assemblages C to G are specific genotypes affecting restricted animal hosts, assemblages A and B parasitise both humans and a number of animal species, and have been considered as having zoonotic potential. The main objective of the current study was to investigate the molecular epidemiology of G. duodenalis in animals and humans in the UK. The current study also evaluated multilocus genotyping and determined the protein changes between assemblages A and B

Vaccination against Toxoplasmosis

The intracellular protozoan parasite Toxoplasma gondii can infect a wide range of animal species, and is one of the main causes of infectious abortion and perinatal mortality in sheep. In humans, the parasite can cause abortion and congenital infection, and fatal disease in immunosuppressed patients. In sheep, toxoplasmosis can be controlled by vaccination with a live, attenuated vaccine. However, since such a vaccine has practical disadvantages and is not acceptable for use in humans, various strategies to develop an effective subunit vaccine have been explored. The major surface antigen of T. gondii, named SAG1, is considered as a promising vaccine candidate. Equally as important as identification of protective antigens is the choice of adjuvant. The immunostimulating complex (iscom) is an adjuvant formulation that induces both humoral and cellular immune responses that are predominantly of type 1, and therefore is likely to be effective against intracellular parasites. The aim of the present study was to produce iscoms containing recombinant SAG1 (rSAG1) and to investigate their immunogenicity and protective capacity against T. gondii using a mouse model. SAG1 expressed in E. coli as a recombinant protein with a hexahistidyl (His6) tag was coupled to preformed iscom matrix (i.e. iscom particles without any antigen) using the affinity of the His6 tag to divalent anions. The matrix contained a chelating lipid and had been loaded with Ni2+ ions. Analytical density gradient centrifugation revealed that a substantial proportion of the SAG1 had bound to the matrix. To investigate the immunogenicity of the rSAG1 iscoms, mice were immunised twice and the cellular immune response examined by in vitro stimulation of spleen cells. Cells from three of four immunised mice proliferated significantly when exposed to rSAG1, whereas cells from only one of five mice were stimulated with T. gondii lysate. ELISA analysis revealed high antibody titres against rSAG1 but only low levels against T. gondii antigens. In two subsequent challenge experiments, three groups of mice were inoculated three times with either rSAG1 iscoms, iscom matrix, or PBS. The third immunisation resulted in substantially higher antibody titres against T. gondii antigen. After inoculation with the virulent RH strain all mice died without any significant differences in survival time between groups (p = 0.179). However, when the mice were inoculated orally with tissue cysts of the Tg-SweF1 strain, significantly lower numbers of brain cysts were found in mice immunised with rSAG1 iscoms than in mice injected with PBS (p < 0.05). In conclusion, although immunisation with rSAG1 iscoms did not protect mice from the lethal challenge infection, partial protection was induced as demonstrated by the reduction of brain cyst load after inoculation with an avirulent strain

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 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

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