A morpho-phylogenetic update on ixodid ticks infesting cattle and buffalos in Vietnam, with three new species to the fauna and a checklist of all species indigenous to the country.

BACKGROUND: Southeast Asia is regarded as a hotspot for the diversity of ixodid ticks. In this geographical region, Vietnam extends through both temperate and tropical climate zones and therefore has a broad range of tick habitats. However, molecular-phylogenetic studies on ixodid tick species have not been reported from this country. METHODS: In this study, 1788 ixodid ticks were collected from cattle, buffalos and a dog at 10 locations in three provinces of northern Vietnam. Tick species were identified morphologically, and representative specimens were molecularly analyzed based on the cytochrome c oxidase subunit I (cox1) and 16S rRNA genes. Fifty-nine tick species that are indigenous in Vietnam were also reviewed in the context of their typical hosts in the region. RESULTS: Most ticks removed from cattle and buffalos were identified as Rhipicephalus microplus, including all developmental stages. Larvae and nymphs were found between January and July but adults until December. Further species identified from cattle were Rhipicephalus linnaei, Rhipicephalus haemaphysaloides, Amblyomma integrum and Haemaphysalis cornigera. Interestingly, the latter three species were represented only by adults, collected in one province: Son La. The dog was infested with nymphs and adults of R. linnaei in July. Phylogenetically, R. microplus from Vietnam belonged to clade A of this species, and R. haemaphysaloides clustered separately from ticks identified under this name in China, Taiwan and Pakistan. Amblyomma integrum from Vietnam belonged to the phylogenetic group of haplotypes of an Amblyomma sp. reported from Myanmar. The separate clustering of H. cornigera from Haemaphysalis shimoga received moderate support. CONCLUSIONS: Three tick species (R. linnaei, A. integrum and H. cornigera) are reported here for the first time in Vietnam, thus increasing the number of indigenous tick species to 62. Clade A of R. microplus and at least R. linnaei from the group of Rhipicephalus sanguineus sensu lato occur in the country. There is multiple phylogenetic evidence that different species might exist among the ticks that are reported under the name R. haemaphysaloides in South and East Asia. This is the first report of A. integrum in Southeastern Asia.

Molecular identification of new Trypanosoma evansi type non-A/B isolates from buffaloes and cattle in Indonesia.

Trypanosoma evansi is reportedly divided into two genotypes: types A and B. The type B is uncommon and reportedly limited to Africa: Kenya Sudan, and Ethiopia. In contrast, type A has been widely reported in Africa, South America, and Asia. However, Trypanosoma evansi type non-A/B has never been reported. Therefore, this study aims to determine the species and genotype of the Trypanozoon subgenus using a robust identification algorithm. Forty-three trypanosoma isolates from Indonesia were identified as Trypanosoma evansi using a molecular identification algorithm. Further identification showed that 39 isolates were type A and 4 isolates were possibly non-A/B types. The PML, AMN-SB1, and STENT3 isolates were likely non-A/B type Trypanosoma evansi isolated from buffalo, while the PDE isolates were isolated from cattle. Cladistic analysis revealed that Indonesian Trypanosoma evansi was divided into seven clusters based on the gRNA-kDNA minicircle gene. Clusters 6 and 7 are each divided into two sub-clusters. The areas with the highest genetic diversity are the provinces of Banten, Central Java (included Yogyakarta), and East Nusa Tenggara. The Central Java (including Yogyakarta) and East Nusa Tenggara provinces, each have four sub-clusters, while Banten has three.

p67 gene alleles sequence analysis reveals Theileria parva parasites associated with East Coast fever and Corridor disease in buffalo from Zambia.

Theileriosis caused by Theileria parva infections is responsible for high cattle mortalities in Zambia. Although infected buffalo are a risk to cattle, the characterization of T. parva parasites occurring in this host in Zambia has not been reported. Furthermore, considering the advances in the development of a p67 subunit vaccine, the knowledge of p67 genetic and antigenic diversity in both cattle and buffalo associated T. parva is crucial. Therefore, blood samples from buffalo (n=43) from Central, Eastern and Southern provinces, and cattle (n=834) from Central, Copperbelt, Eastern, Lusaka, and Southern provinces, were tested for T. parva infection and the parasites characterized by sequencing the gene encoding the p67 antigen. About 76.7 % of buffalo and 19.3 % of cattle samples were PCR positive for T. parva. Three of the four known p67 allele types (1, 2 and 3) were identified in parasites from buffalo, of which two (allele types 2 and 3) are associated with T. parva parasites responsible for Corridor disease. Only allele type 1, associated with East Coast fever, was identified from cattle samples, consistent with previous reports from Zambia. Phylogenetic analysis revealed segregation between allele type 1 sequences from cattle and buffalo samples as they grouped separately within the same sub-clade. The high occurrence of T. parva infection in buffalo samples investigated demonstrates the risk of Corridor disease infection, or even outbreaks, should naïve cattle co-graze with infected buffalo in the presence of the tick vector. In view of a subunit vaccine, the antigenic diversity in buffalo associated T. parva should be considered to ensure broad protection. The current disease control measures in Zambia may require re-evaluation to ensure that cattle are protected against buffalo-derived T. parva infections. Parasite stocks used in 'infection and treatment' immunization in Zambia, have not been evaluated for protection against buffalo-derived T. parva parasites currently circulating in the buffalo population.

Detection of Toxoplasma gondii infection in buffaloes (Bubalus bubalis) and cattle (Bos taurus) at the Tabriz abattoir, Iran.

BACKGROUND: Toxoplasma gondii is a widely prevalent zoonotic protozoan parasite in humans and warm-blooded animals worldwide. Infection of humans by this parasite can result in severe clinical symptoms, particularly in individuals with congenital toxoplasmosis or immunocompromised patients. Contamination mainly occurs through foodborne routes, especially the consumption of raw or undercooked meat from animals. OBJECTIVES: The aim of this study was to use PCR to detect T. gondii in tissues and organs of buffaloes and cattle slaughtered at Tabriz slaughterhouse, in Iran. METHODS: Fifty grams of heart, thigh, diaphragm and tongue from 50 buffaloes and 100 cattle slaughtered at the Tabriz industrial slaughterhouse were selected for sampling using a combination of convenience sampling. The samples were tested using a previously published PCR method. RESULTS: Out of the 150 animal samples, T. gondii was detected in 10 (6.7%, 95%CI: 3.2-11.9), including one buffalo (2%, 95%CI: 0.1-10.6) and nine cattle (9%, 95%CI: 4.2-16.4). There was no statistically significant difference in the rate of T. gondii infection among cattle based on age and sex (p > 0.05). CONCLUSIONS: The results indicated a potential risk of T. gondii transmission to humans through the consumption of infected meat. Therefore, appropriate and effective preventive measures should be taken to limit the transmission of this parasite to humans, and the consumption of raw and undercooked meat should be discouraged.

Prevalence and clinico-pathology of PSOROPTIC mange in buffaloes of the Amazon region.

Scabies is an important skin disease in several species of domestic and wild animals; however, few reports in Brazil have emphasized its occurrence in buffaloes. This article describes the epidemiological, clinical and pathological aspects and diagnosis of psoroptic mange in buffaloes in a property in the municipality of Castanhal, PA, Amazon region. Of the 41 buffaloes examined, 38 males and females of the Murrah, Baio, Mediterranean and Carabao breeds and their crossbreeds, aged between 2 and 20 years, had a history of pruritus. Clinical examination was performed to map the lesions, skin scrapings were collected to identify the mites, and a biopsy was performed for histopathological examination. Clinical signs, from mild to severe intensity, varied according to the system of creation and handling of the animals and were more severe in buffaloes raised in bays than those raised under a collective regime (pastures and collective troughs). The characteristic clinical signs were intense itching, extensive areas of alopecia, periocular edema, and thickening of the epidermis with exudative crusts covering the face, chamfer, neck, scapular region, back, base of the horn, thoracic and pelvic limbs and chest. The behavior of rubbing the affected regions of the body against structures (troughs, fence posts, gates) or with the horns was frequently observed and provided relief from itching. In the most severe cases, mites were also noted in the crusts, which were identified as Psoroptes natalensis. Histological skin lesions exhibited alterations consistent with immune-mediated dermatitis, which is typical of hypersensitivity to mite-derived allergens.

Investigating the infection dynamics and molecular detection of Cryptosporidium in Buffaloes in Sylhet, Bangladesh.

Cryptosporidiosis, a zoonotic infection impacting both livestock and humans, is inadequately understood in terms of its prevalence and transmission dynamics involving buffaloes in Bangladesh. This research, conducted in the Sylhet division, aimed to explore the prevalence and potential risk factors influencing Cryptosporidium spp. in the faecal samples of 392 buffaloes. Detection of the parasite utilized modified Ziehl-Neelsen staining, with molecular identification achieved through nested PCR (nPCR). The comprehensive analysis revealed 9.18% (36/392) prevalence at the individual animal level and 40.48% (17/42) at the herd level. Age-based analysis revealed fluctuating infection rates of Cryptosporidium spp. in buffaloes across distinct age brackets, with rates of 22.61% in those aged 0-6 months, 5.00% in those aged 6-12 months, and 1.03% in those aged 12-18 months. Diarrheic buffaloes showed a significantly (p < 0.001) higher infection rate (26.67%; 28/105) compared to non-diarrheic buffaloes (2.79%; 8/287). In risk factor analysis, binary logistic regression revealed that buffaloes aged 0-6 months were experiencing a likelihood that is 14.84 times higher to be affected by Cryptosporidium in contrast to their older counterparts (OR = 14.85; p = 0.02). Additionally, diarrhoeic buffaloes were found to be more susceptible to Cryptosporidium compared to healthy buffaloes (OR = 17.50; p < 0.001). A higher stocking density was associated with an increased likelihood of infection in buffaloes (OR = 11.20; p = 0.01). The results of this study emphasize the necessity for targeted interventions, considering factors like diarrheic condition and stocking density, to effectively manage and control cryptosporidiosis in Bangladesh.

Clinico-haematobiochemical and cardiac alterations in Trypanosoma evansi infected buffaloes of Andhra Pradesh, India.

The present research aimed to document the incidence, clinical signs, haematological, and serum biochemical alterations, as well as electrocardiography and echocardiography findings in 62 buffaloes (selected from a total of 240) infected with Trypanosoma evansi. The study spanned one year, from January 2022 to December 2022. Morphological identification of Trypanosoma evansi was done by the presence of a centrally positioned nucleus with a small sub-terminal kinetoplast at the posterior position through microscopic examination of Giemsa stained peripheral blood smears. The incidence of trypanosomosis were determined to be 26% (62/240) using stained blood smear examination and 41% (98/240) through polymerase chain reaction assay. Clinical signs exhibited by buffaloes with trypanosomosis included the lack of rumination (94%; 58/62), anorexia (90%; 56/62), emaciation (87%; 54/62), loss of milk yield (84%; 52/62), ocular discharges (82%; 51/62), depressed demeanour (81%; 50/62), sunken eye balls (61%; 38/62), fever (60%; 37/62), scleral congestion (56%; 35/62) and intermittent fever (42%; 26/62). Cardiovascular clinical findings in affected buffaloes included tachycardia (44%; 27/62), cardiac arrhythmia (24%; 15/62), cardiac murmurs (19%; 12/62) and muffled heart sounds (18%; 11/62). In the present study, buffaloes with trypanosomosis exhibited significant reduction in haemoglobin (p = 0.008), packed cell volume (p = 0.004), total erythrocyte count (p = 0.003), mean corpuscular volume (p = 0.042), total leucocyte count (p = 0.048) and absolute neutrophil count (p = 0.012); a significant increase in absolute eosinophil count (p = 0.011) and absolute monocyte count (p = 0.008) compared to the apparently healthy buffaloes. Additionally significant decrease in albumin (p = 0.001), A/G ratio (p = 0.007), calcium (p = 0.008), glucose (p = 0.007), phosphorous (p = 0.048), sodium (p = 0.008), potassium (p = 0.041) and chloride (p = 0.046) were observed in buffaloes with trypanosomosis compared to healthy ones. Buffaloes with trypanosomosis also showed significant increase in globulin (p = 0.004), aspartate aminotransferase (p = 0.008), bilirubin (p = 0.034), blood urea nitrogen (p = 0.071), creatinine (p = 0.029), cholesterol (p = 0.046), lactate dehydrogenase (p = 0.009), gamma-glutamyl transferase (p = 0.004) and creatine kinase-myoglobin binding levels (p = 0.005). Electrocardiography explorations in buffaloes with trypanosomosis revealed sinus tachycardia, low voltage QRS complex, ST segment elevation, wide QRS complex, sinus arrhythmia, sinus bradycardia, wandering pace maker, first degree atrio ventricular block, biphasic T wave and tall T wave. Echocardiography examination unveiled cardiac chamber dilatation, ventricular wall thickening and indications of pericarditis/cardiac tamponade. Necropsy was carried on the dead buffaloes during the study period disclosed severely congested blood vessels on epicardial surface, endocardial haemorrhages, and presence of pericardial fluid. Histopathological examination of the heart revealed hyaline degeneration, haemorrhages in the cardiac muscles and varying degrees of degenerative changes. Additionally, the pericardium displayed increased thickness due to presence of more elastic fibres, fibroblast cells in the myocardium, discontinuity of muscle layers, vascular congestion, perivascular mono nuclear cell infiltration and augmented thickness of the endocardium with fibroblast cell proliferation. The study's conclusion highlights cardiac alterations as secondary complications in buffaloes infected with Trypanosoma evansi. Further investigations are recommended to elucidate therapeutic modifications and refine the treatment paradigm.

Epidemiology of rumen fluke infection in selected buffalo farms in perak, malaysia: prevalence, molecular species identification, and associated risk factors.

Rumen flukes cause heavy economic losses in the ruminant industry worldwide, especially in tropical and subtropical countries. This study estimated the prevalence of rumen flukes in buffaloes, identified the species diversity, and determined risk factors associated with rumen fluke prevalence in Perak, Peninsular Malaysia. A cross-sectional study was conducted, and 321 faecal samples were collected from six buffalo farms. A structured questionnaire was developed, and farmers were interviewed to obtain information regarding risk factors associated with rumen fluke infection. The faecal samples were examined using sedimentation and Flukefinder® techniques. Genomic DNA was extracted from the fluke eggs recovered using the Flukefinder® method, and the internal transcribed spacer 2 (ITS2) fragment was amplified and sequenced to facilitate species identification. The results showed that the overall prevalence of rumen fluke across the sampled farms was 40.2% (129/321). Three rumen fluke species were identified, namely, Fischoederius elongatus, F. cobboldi, and Orthocoelium streptocoelium. Several management factors had a significant association (P < 0.05) with rumen fluke prevalence, including production type, cleaning of the stable, drinking water system, flooding around the farm, grazing system, pasture sharing with other livestock, and deworming program. This work constitutes the first attempt to understand the epidemiology of rumen fluke infection in the region and suggests that good farm management, pasture management, choosing appropriate drugs, and proper husbandry practices may improve buffalo health and production in areas where rumen flukes are prevalent.

Diversity, prevalence and risk factors associated to gastrointestinal tract parasites in wild and domestic animals from Pakistan.

The gastrointestinal tract (GIT) of animals contains various helminth parasites, responsible for subclinical and clinical parasitism. The health situation of animals is seriously affected by these parasites and leads massive financial losses for the livestock trade. The present study was conducted to determine the prevalence and associated risk factors of GIT parasites in small and large ruminants of Mardan district and zoo animals of Golden Life Safari Park Khyber Pakhtunkhwa. A total of 419 fecal samples were collected including 117 from goats, 110 from cattle, 80 from sheep, 66 from buffaloes, 40 from birds, four from deer, and two from leopards. The fecal samples were subjected to sedimentation and floatation to identify parasite eggs. Of a total of 419 fecal samples, 215 (51.31%) were found positive for GIT parasites eggs. The prevalence of GIT parasites was high in sheep (65%), followed by goats (62.39%), cattle (42.70%), and buffaloes (37.87%). In birds, GIT parasite eggs were identified in 11 (45.83%) and 7 (43.75%) fecal samples of Pheasant and Peacock, respectively. However, an absence of infection was noted in fecal samples from deer and leopards. Eggs of GIT Parasites belonging to one superfamily and four different genera were recognized in fecal samples of domestic animals and buffaloes, which were Trichostrongyloidea (19.81%), Strongyloides (9.31%), Trichuris (7.77%), Monezia (6.44%), and Fasciola (4.53%). For bird, the coccidia group comprises the highest representation of parasites (2.63%), followed by the Trichuris genus (1.67%). Settlement, management system, anthelminthic medications and body condition score were significantly associated with the occurrence of GIT parasites in feces (P < 0.05). Animals from rural settlements, rearing under poor management systems, with thin (poor) body condition and under warmed were more affected by GIT parasites. The diversity and the high prevalence of GIT parasites reported in this study highlights the urgent need for intervention through treatment and modification of management practices in the investigated areas of Pakistan.

Molecular Detection of Animal Trypanosomes in Different Animal Species in the Visayas Region of the Philippines.

PURPOSE: Animal trypanosomosis is one of the most important parasitic diseases significantly affecting the Philippine economy. It is considered by the government to be the second most important disease of livestock after fasciolosis. A PCR-based molecular survey for trypanosomes in different animals in Bohol, Philippines, was performed to assess the prevalence of trypanosomosis in the area during the rainy and dry season. METHODS: A total of 269 blood samples were collected in two batches in rainy and dry season from different animal species in Ubay Stock Farm in Ubay, Bohol, the Philippines, including 151 samples from water buffaloes, 76 samples from cattle, 35 samples from goats, and 7 samples from horses. DNA was subsequently extracted from these blood samples, and two different PCR assays were employed to detect and identify trypanosomes DNA including ITS1 PCR and CatL PCR. RESULTS: Animal trypanosomes, Trypanosoma evansi and Trypanosoma theileri, were detected in water buffalo (37.7%) [95%CI: 30.4 - 45.7], cattle (44.7%) [95%CI: 34.1 - 55.9], and goats (34.3%) [95%CI: 20.8 - 50.8]. Only T. evansi was detected in horses (28.6%) [95% CI: 8.2 - 64.1]. No clinical signs were observed in all positive animals. CONCLUSION: This highlights the importance of domestic animals that can be infected with no signs but may act as reservoir animals and transmit trypanosomosis to susceptible animals. This study supports the importance of regular surveillance to estimate the prevalence of the disease, emphasizing its various dynamics in the affected areas and supporting efficient intervention measures.

Distribution of Toxocara vitulorum in Cattle of Agri Region

OBJECTIVE: Parasitizing in the small intestines of cattle, buffalo and bison, Toxocara vitulorum is seen in countries with humid tropical and subtropical climates, including Türkiye. Adult parasites can cause digestive disorders such as loss of appetite, weakness, diarrhea or constipation, and sometimes death, especially in calves. This study was planned to investigate the distribution of T. vitulorum in cattle of the Agri region. METHODS: In this study, stool samples of the size of a walnut were taken from the rectum of 200 calves 0-6 months old and 200 cattle older than 1 year in the province of Agri and its districts, and the preparations were prepared using the saturated zinc sulfate flotation method and the preparations were examined under a light microscope. RESULTS: T. vitulorum eggs were detected in 70 (35%) of 200 calves aged 0-6 months and 21 (10.5%) of 200 cattle over one year old. The difference in positivity between cattle over one year old and calves was statistically significant (p=0.001). With fecal examination in 22 of the calves (11%), Trichostrongylidae spp. eggs were detected. CONCLUSION: It was determined that the prevalence of T. vitulorum in calves was high in Diyadin, Eleskirt, Dogubayazit, Hamur, Tasliçay, Tutak and Patnos districts of Agri province. We believe that larger-scale studies should be conducted on the spread of this parasite, which causes significant low yields in cattle breeding.

The determination and relationship of four coexisting paramphistomes in perspective of integrative taxonomic investigation.

Co-infections with Orthocoelium species and other paramphistomes were found in different ruminant hosts from two provinces of Thailand. Whilst O. parvipapillatum coexisted with Paramphistomum epiclitum in the same cattle (Bos taurus) from Pathum Thani Province, Thailand, O. dicranocoelium and Fischoederius elongatus were found in water buffalo (Bubalus bubalis) from Chiang Mai Province. Morphological, histological, and tegumental surface features of both Orthocoelium species were intensively investigated for species differentiation. Statistical analysis of eight morphometric ratios presented morphological differences for three paramphistomes in the Paramphistomidae family and some relationships among paramphistomes in different definitive hosts. The genetic relationships of the co-infecting paramphistomes were investigated using p-distance and phylogenetic tree analyses. Genetic variations in the Orthocoelium co-infecting paramphistomes, P. epiclitum and F. elongatus, were calculated and compared to DNA sequence alignments based on internal transcribed spacer 2 (ITS2) and cytochrome c oxidase subunit I (COI) DNA markers. In addition, the phylogenetic tree constructions from both DNA markers and their concatenated sequence (ITS2 + COI) were used for species confirmation and the presentation of genetic relationships between co-infecting paramphistomes and other paramphistomes. This study improves the basic taxonomical description and understanding of parasite-parasite and host-parasite interactions from the perspectives of morpho-histological, morphometric, and genetic variation in co-infecting paramphistomes and Orthocoelium species in different hosts.

Conservation and variation in the region of the Theileria parva p104 antigen coding gene used for PCR surveillance of the parasite.

The range of the protozoan parasite Theileria parva, which causes East Coast fever in cattle, has been expanding to countries where it has not previously been detected, as a result of cross-border domestic cattle movement. Countries where T. parva has not previously been observed until recently include Cameroon and South Sudan. This raises the issue of the conservation of the p104 antigen gene, on which the nested PCR assay that is widely used for T. parva surveillance in the blood of infected cattle is based. We sampled 40 isolates from six countries widely distributed across the geographical range of the parasite, including eastern, central and southern Africa, for p104 sequence polymorphism. These included parasites from both domestic cattle and the Cape buffalo (Syncerus caffer) wildlife reservoir. The most frequent allelic variants were present in cattle transmissible isolates from multiple widely separated geographical regions in Zambia, Uganda, Kenya, Tanzania, Rwanda and South Africa. These frequent p104 variants were also present in the three component stocks of the Muguga cocktail used for the infection and treatment live immunisation procedure to control T. parva in the field. Other isolates exhibited unique alleles. This includes some of the p104 sequences from Cameroon, which is outside the known range of the Rhipicephalus tick vector and whose origin is therefore unclear. The nested primer oligonucleotides used to generate the amplicons were universally conserved in cattle-derived parasites and a majority of buffalo-derived isolates across the geographical range of the parasite. However, some rare South African buffalo-derived isolates exhibited one or two mismatches with the primer sequences. It therefore remains possible that some p104 alleles may be so divergent that they do not amplify with the current diagnostic primers and are not detectable in surveys, hence the need for increasing knowledge of genetic heterogeneity of diagnostic targets. There was no evidence for positive selection among those p104 mutations that resulted in residue changes. Importantly, the data indicate that the p104-based PCR detection assay should be effective across the majority of the range of T. parva, and if the one or two mismatches are shown in future to result in the primers annealing less efficiently, then the assay can be further improved by introduction of degenerate bases to enable amplification of the less frequent South African buffalo-derived variant p104 genes.

Phylogenetic characterization and analysis of Sarcocystis buffalonis.

Despite its frequent presence in buffaloes, Sarcocystis buffalonis remains as one of the most under studied parasite. In the present study, isolates of S. buffalonis from,Mathura, Uttar Pradesh India were characterized for 18S rRNA (MF595842-MF595844), cox 1 (MG792800-MG792802), 28S rRNA (MH793418-MH793420) and ITS 1 (MH793421-MH793423) genes. Analysis revealed multiple haplotypes for each individual gene viz., 18S rRNA (three haplotypes), cox 1 (two haplotypes), 28S rRNA (two haplotypes) and ITS 1 (single haplotype). The studied Indian sequences showed variable homologies for individual gene loci viz., 18S rRNA (99.3-99.9%); cox 1 (99.8-100.0%); 28S rRNA (99.9-100.0%) and ITS 1 (100.0%) The phylogenetic association between S. buffalonis and closely related Sarcocystis spp. infecting buffaloes and cattle was delineated. All these gene loci placed S. buffalonis close to S. hirsuta. The study has generated A vital phylogenetic data about this erstwhile neglected parasite.

Intestinal parasites of buffalo calves from Romania: molecular characterisation of Cryptosporidium spp. and Giardia duodenalis, and the first report of Eimeria bareillyi.

Buffaloes represent an important economic resource for several regions of the world including Romania. In the present study, we examined 104 faecal samples collected from 38 buffalo calves (2-11 weeks old) from household rearing systems in Romania for gastrointestinal parasites. All samples were tested using the saturated salt flotation, McMaster and modified Ziehl-Nielsen staining methods. PCR coupled with sequencing isolates were used to identify assemblages of Giardia lamblia (Kunstler, 1882) and species of Cryptosporidium Tyzzer, 1907. Overall, 33 out of 38 examined buffalo calves were infected with different gastrointestinal parasites: 16 had single infections and 17 had mixed infections with two or three parasites. Species of Eimeria Schneider, 1875 (32/38; 84%) were the most prevalent parasites; eight species were identified according to the oocyst morphology, including the pathogenic E. bareillyi (Gill, Chhabra et Lall, 1963) which was detected for the first time in buffaloes from Romania. The nematodes Toxocara vitulorum (Goeze, 1782) (11/38; 37%) and Strongyloides papillosus (Wedl, 1856) (6/38; 16%) were also detected. Cryptosporidium spp. were found in four (11%) buffalo calves; two of them were molecularly identified as C. ryanae Fayer, Santin et Trout, 2008, and another one clustered in the same clade with C. ryanae, C. bovis Fayer, Santin et Xiao, 2005, and C. xiaoi Fayer et Santin, 2009. Giardia duodenalis assemblage E was also molecularly detected in a single (2.6%) buffalo calf. The presence of other buffaloes in the same barn was identified as a risk factor for infection with T. vitulorum. Our results indicate extensive parasitic infections in buffalo calves from northwestern Romania and underline the necessity of prophylactic treatments for T. vitulorum and E. bareillyi.

Transcriptomic landscape of hepatic lymph nodes, peripheral blood lymphocytes and spleen of swamp buffaloes infected with the tropical liver fluke Fasciola gigantica.

The tropical liver fluke Fasciola gigantica is a parasitic helminth that has been frequently reported to infect mammals, typically involving water buffaloes. In this study, we characterized the tissue transcriptional landscape of buffaloes following infection by F. gigantica. RNAs were isolated from hepatic lymph nodes (hLNs), peripheral blood lymphocytes (pBLs), and spleen at 3-, 42- and 70-days post-infection (dpi), and all samples were subjected to RNA sequencing analyses. At 3 dpi, 2603, 460, and 162 differentially expressed transcripts (DETs) were detected in hLNs, pBLs, and spleen, respectively. At 42 dpi, 322, 937, and 196 DETs were detected in hLNs, pBLs, and spleen, respectively. At 70 dpi, 376, 334, and 165 DETs were detected in hLNs, pBLs, and spleen, respectively. Functional enrichment analysis identified upregulated immune-related pathways in the infected tissues involved in innate and adaptive immune responses, especially in hLNs at 42 and 70 dpi, and pBLs at 3 and 42 dpi. The upregulated transcripts in spleen were not enriched in any immune-related pathway. Co-expression network analysis further identified transcriptional changes associated with immune response to F. gigantica infection. Receiver operating characteristic (ROC) curve analysis showed that 107 genes in hLNs, 32 genes in pBLs, and 36 genes in spleen correlated with F. gigantica load. These findings provide new insight into molecular mechanisms and signaling pathways associated with F. gigantica infection in buffaloes.

Molecular survey of Toxoplasma gondii in cattle and buffaloes and phylogenetic position of Pakistani isolates based on ITS-1 gene.

Toxoplasma gondii (T. gondii) is a facultative heterogeneous parasite that belongs to Apicomplexa and can infect almost all warm-blooded animals, including ruminants, birds and humans. To date, no information is available about the molecular investigation of T. gondii in large ruminants from Pakistan. In the present study, prevalence, risk factors and genetic diversity of this parasite were evaluated by using PCR based on ITS-1 gene followed by sequencing of three selected positive PCR products. A total of 310 blood samples from cattle (N = 190) and buffaloes (N = 120) were collected from randomly selected farms located in Rajanpur district in Punjab (Pakistan). The overall infection rates of T. gondii were 12.2% (23/190) and 0% (0/120), respectively, in cattle and buffaloes. All studied epidemiological factors were not found associated with T. gondii infection in cattle. Sequence analysis of our T. gondii isolates infecting cattle revealed only one sequence considered as the most represented genetic variant (GV1) among T. gondii isolates around the world. Phylogenetic analysis revealed that ITS-1 partial sequences of our isolates clustered with those from T. gondii isolates infecting goats and birds from Pakistan and other isolates found in several animal species from different worldwide countries like China, Thailand, Poland, Canada, USA and Brazil. Our report indicates a natural infection with T. gondii of cattle for the first time in Pakistan by using molecular method. This study is important to the design of control strategy against this parasite in order to improve the output of livestock sector which is the main income source of the population in Pakistan.

Prevalence and risk factors of Toxocara vitulorum infection in buffalo calves in coastal, northeastern and northwestern regions of Bangladesh.

Buffalo (Bubalus bubalis) is popularly known as the black gold of South Asia, consisting of 97% of the world buffalo population. Among the parasitic infections, Toxocara vitulorum is one of the most common and harmful parasites of buffalo calves in Bangladesh. A cross-sectional study was conducted to explore the prevalence and associated risk factors of T. vitulorum infection of buffalo calves in four regions of Bangladesh. A total of 1751 fecal samples were collected and examined using flotation followed by the McMaster technique for counting the eggs per gram of feces (EPG) of T. vitulorum. The overall prevalence of T. vitulorum infection in buffalo calves was 22.9%. Significantly (p < 0.001) higher prevalence was found in the Barishal coastal area (35.7%) followed by Chattogram coastal area (29.2%), northeastern region (15.5%) and northwestern region (8.3%). Buffalo calves aged 1-3 months were heavily infected with T. vitulorum (51.7%) which was statistically different (p < 0.001) compared to those >3-6 months (27.6%) and > 6-12 months (6.5%). According to univariate analysis, coastal regions, rainy season, young age, gender, indigenous river type, buffalo calves with poor body condition and soft feces were found significantly associated with T. vitulorum infections. Coastal regions, rainy season and young age were identified as the risk factors of T. vitulorum infection in buffalo calves by final logistic regression model. This study reveals that T. vitulorum infection is endemic in Bangladesh and widely distributed in the coastal regions. Therefore, attention to buffalo calves regarding in-depth clinical effects and current therapeutic approaches against this nematode should be evaluated along with the economic impact of infection. Exploring the genetic diversity of T. vitulorum may help to reveal the host-parasite relationship in the future.

Extracellular vesicles from Fasciola gigantica induce cellular response to stress of host cells.

Extracellular vesicles (EVs) from parasitic helminths play an important role in immunomodulation. However, EVs are little studied in the important parasite Fasciola gigantica. Here the ability of EVs from F. gigantica to induce cellular response to stress (reactive oxygen species generation, autophage and DNA damage response) in human intrahepatic biliary epithelial cells (HIBEC) was investigated. F. gigantica-derived EVs were isolated by ultracentrifugation, and identified with transmission electron microscopy, nanoparticle size analysis and parasite-derived EV markers. Internalization of EVs by HIBEC was determined by confocal immunofluorescence microscopy and flow cytometry. ROS levels in HIBEC were detected by molecular probing. EVs-induced autophagy and DNA-damaging effects were determined by evaluating expression levels of light chain 3B protein (LC3B), phosphor- H2A.X and phosphor-Chk1, respectively. Results revealed that EVs with sizes predominately ranging from 39 to 110 nm in diameter were abundant in adult F. gigantica and contained the parasite-derived marker proteins enolase and 14-3-3, and EVs were internalized by HIBEC. Further, uptake of EVs into HIBEC was associated with increased levels of reactive oxygen species, LC3Ⅱ, phosphor-H2A.X and phosphor-Chk1, suggesting EVs are likely to induce autophagy and DNA damage & repair processes. These results indicate F. gigantica EVs are associated with modulations of host cell responses and have a potential important role in the host-parasite interactions.

High-quality reference genome of Fasciola gigantica: Insights into the genomic signatures of transposon-mediated evolution and specific parasitic adaption in tropical regions.

Fasciola gigantica and Fasciola hepatica are causative pathogens of fascioliasis, with the widest latitudinal, longitudinal, and altitudinal distribution; however, among parasites, they have the largest sequenced genomes, hindering genomic research. In the present study, we used various sequencing and assembly technologies to generate a new high-quality Fasciola gigantica reference genome. We improved the integration of gene structure prediction, and identified two independent transposable element expansion events contributing to (1) the speciation between Fasciola and Fasciolopsis during the Cretaceous-Paleogene boundary mass extinction, and (2) the habitat switch to the liver during the Paleocene-Eocene Thermal Maximum, accompanied by gene length increment. Long interspersed element (LINE) duplication contributed to the second transposon-mediated alteration, showing an obvious trend of insertion into gene regions, regardless of strong purifying effect. Gene ontology analysis of genes with long LINE insertions identified membrane-associated and vesicle secretion process proteins, further implicating the functional alteration of the gene network. We identified 852 predicted excretory/secretory proteins and 3300 protein-protein interactions between Fasciola gigantica and its host. Among them, copper/zinc superoxide dismutase genes, with specific gene copy number variations, might play a central role in the phase I detoxification process. Analysis of 559 single-copy orthologs suggested that Fasciola gigantica and Fasciola hepatica diverged at 11.8 Ma near the Middle and Late Miocene Epoch boundary. We identified 98 rapidly evolving gene families, including actin and aquaporin, which might explain the large body size and the parasitic adaptive character resulting in these liver flukes becoming epidemic in tropical and subtropical regions.