Molecular Epidemiology and Characterization of Theileria in Goats.

The study was carried out to determine the prevalence and associated risk factors of theileriosis in goats of Chattogram district, Bangladesh. Molecular characterization of circulating Theileria in this area was also undertaken. A total of 400 samples were collected from goats of different breeds, ages and sex with relevant information of rearing and management. The prevalence of theileriosis was 8.50% (34/400) by polymerase chain reaction though all of those samples were test-negative by microscopic examination. Among different risk factors season, breed and tick infestation were found to be significantly (p ≤ 0.05) associated with the prevalence of theileriosis in goats. Serous nasal discharge and swollen lymph nodes were determined to be significant clinical signs. The Theileria spp. detected in the present study closely resemble isolates which were previously detected in Myanmar and China. Further large scale epidemiological studies are required to identify the circulating species and responsible vectors, which would facilitate control measures for this disease in Bangladesh.

An appraisal of oriental theileriosis and the Theileria orientalis complex, with an emphasis on diagnosis and genetic characterisation.

Oriental theileriosis, a tick-borne disease of bovids caused by members of the Theileria orientalis complex, has a worldwide distribution. Globally, at least 11 distinct genotypes of T. orientalis complex, including type 1 (chitose), type 2 (ikeda), type 3 (buffeli), types 4 to 8, and N1-N3, have been described based on the sequence of the major piroplasm surface protein (MPSP) gene. Of these 11 genotypes, mainly ikeda and chitose are known to be pathogenic and cause considerable morbidity (including high fever, anaemia, jaundice and abortion), production losses and/or mortality in cattle. Mixed infections with two or more genotypes of T. orientalis is common, but do not always lead to a clinical disease, posing challenges in the diagnosis of asymptomatic or subclinical forms of oriental theileriosis. The diagnosis of oriental theileriosis is usually based on clinical signs, the detection of piroplasms of T. orientalis in blood smears, and/or the use of serological or molecular techniques. This paper reviews current methods used for the diagnosis of T. orientalis infections and the genetic characterisation of members of the T. orientalis complex, and proposes that advanced genomic tools should be established for investigations of these and related haemoparasites.

Multiplexed-tandem PCR (MT-PCR) assay to detect and differentiate gastrointestinal nematodes of alpacas.

BACKGROUND: Gastrointestinal nematodes (GINs) frequently infect South American camelids (alpacas and llamas) and cause economic losses due to reduced production of fiber, meat and/or leather. Our knowledge about the epidemiology and diagnosis of GINs in llamas and alpacas is limited, and reliable keys for the identification of the third-stage larvae (L3s) of some common nematodes (such as Camelostrogylus mentulatus) that infect alpacas and llamas remain undescribed. In this study, we modified two existing semi-quantitative multiplexed-tandem (MT)-PCR assays, originally developed for the GINs of sheep and cattle, to reliably detect and differentiate the common genera/species of GINs in the faeces of alpacas. RESULTS: Following the establishment of the MT-PCR assay using positive and negative control samples, alpaca faecal samples were tested to validate the assay to detect and differentiate nematode genera/species, including C. mentulatus, Cooperia spp., Haemonchus spp., Oesophagostomum spp., Ostertagia ostertagi, Teladorsagia circumcincta and Trichostrongylus spp. Sequencing of the MT-PCR products demonstrated specific (100%) amplification of the target nematode genera/species. Additionally, a comparison of results of the MT-PCR assay and the morphological identification of adult worms collected from the same 35 alpacas revealed that there was a good agreement (37-94%) between the two methods. However, some discrepancies were observed between the results of the MT-PCR assay and the morphological identification of adult worms. CONCLUSIONS: The MT-PCR platform is an accurate, sensitive and rapid method for the diagnosis of GINs in alpacas, and it can be used as a substitute to larval culture to identify common nematodes in the faeces of alpacas and llamas.

Assessing the performance of multiplexed tandem PCR for the diagnosis of pathogenic genotypes of Theileria orientalis using pooled blood samples from cattle.

Oriental theileriosis caused by multiple genotypes of Theileria orientalis is an important tick-borne disease of bovines. Here, we assessed the performance of an established multiplexed tandem PCR (MT-PCR) for the diagnosis of the two recognized, pathogenic genotypes (chitose and ikeda) of T. orientalis in cattle using pooled blood samples. We used a total of 265 cattle blood samples, which were divided into two groups according to previous MT-PCR results for individual samples. Samples in group 1 (n = 155) were from a herd with a relatively high prevalence of T. orientalis infection; and those in group 2 (n = 110) were from four herds with a low prevalence. For group 1, 31 and 15 batches of five- and ten-pooled samples (selected at random), respectively, were formed. For group 2, 22 and 11 batches of five- and ten-pooled samples (selected at random), respectively, were formed. DNAs from individual pooled samples in each batch and group were then tested by MT-PCR. For group 1, the apparent prevalences estimated using the 31 batches of five-pooled samples (97%) and 15 batches of ten-pooled samples (100%) were significantly higher compared with individual samples (75%). For group 2, higher apparent prevalences (9% and 36%) were also recorded for the 22 and 11 batches of pooled samples, respectively, compared with individual samples (7%). Overall, the average infection intensity recorded for the genotypes of chitose and ikeda were considerably lower in pooled compared with individual samples. The diagnostic specificities of MT-PCR were estimated at 95% and 94%, respectively, when batches of five- and ten-pooled samples were tested, and 94% for individual samples. The diagnostic sensitivity of this assay was estimated at 98% same for all individual, five- and ten-pooled samples. This study shows that screening batches of five- and ten-pooled blood samples from cattle herds are similar to those obtained for individual samples, and, importantly, that the reduced cost for the testing of pooled samples represents a considerable saving to herd managers.

An outbreak of oriental theileriosis in dairy cattle imported to Vietnam from Australia.

This study reports an outbreak of oriental theileriosis in dairy cattle imported to Vietnam from Australia. Following clinical and pathological diagnoses, a total of 112 cattle blood samples were divided into three groups and tested using multiplexed tandem PCR. Group 1 were from aborted heifers in Vietnam; group 2 were from cattle before shipment from group 1 cattle and group 3 were from the same batch of cattle but transported to Taiwan. Theileria orientalis DNA was detected in 72·3% cattle. The prevalences of T. orientalis in groups 1, 2 and 3 were 77·6, 86·9 and 57·5%, respectively, and the difference in prevalence was significant between groups 1 and 3 (P < 0·0001). The infection intensities of genotypes chitose and ikeda of T. orientalis were higher in groups 1 (57 721 and 33 709, respectively) and 3 (5897 and 61 766, respectively) than those in group 2 (2071 and 6331, respectively). Phylogenetic analyses of the major piroplasm surface protein sequences revealed that genotypes chitose and ikeda determined herein were closely related to those previously reported from Australia. This first report of an outbreak of oriental theileriosis in imported cattle emphasizes improved measures for the export and import of cattle infected with T. orientalis.

Molecular characterisation of Theileria orientalis in imported and native bovines from Pakistan.

The epidemiological aspects of Theileria orientalis in Pakistan are unknown; therefore, investigations using sensitive and precise molecular techniques are required. This study reports the first molecular characterisation of T. orientalis detected from imported (Bos taurus) and native cattle (Bos indicus×Bos taurus) and buffaloes (Bubalus bubalis) selected from four districts of Punjab, Pakistan. DNA samples from blood (n=246) were extracted and tested using conventional PCR utilising the major piroplasm surface protein (MPSP) gene and multiplexed tandem PCR (MT-PCR). Theileria orientalis DNA was detected (15%; 22/147) only in imported cattle by conventional PCR, whereas 24.5% (36/147), 6% (3/50) and 6.1% (3/49) of the imported cattle and native Pakistani cattle and buffaloes, respectively were test-positive for T. orientalis using MT-PCR. Using MT-PCR, the prevalence of T. orientalis was significantly higher (P<0.0001) in imported cattle compared to that of detected in native Pakistani bovines. The prevalence of T. orientalis and DNA copies of chitose and ikeda were significantly higher (P<0.05) in imported cattle than those detected in native Pakistani bovines. DNA sequencing of amplicons of the conventional PCR revealed the presence of buffeli, chitose and ikeda genotypes of T. orientalis. Phylogenetic analysis revealed that the MPSP sequences of buffeli, chitose and ikeda from imported cattle were closely related to those sequences reported previously from Australia and other regions. This study provides the first survey of T. orientalis infection in imported and native bovines in Pakistan, and highlights the need for future studies to understand the spread of transboundary animal diseases.

Molecular characterization of Theileria orientalis from cattle in Ethiopia.

This study reports the first molecular characterization of Theileria orientalis in local breeds of cattle in Ethiopia. A conventional PCR utilizing major piroplasm surface protein (MPSP) gene and an established multiplexed tandem PCR (MT-PCR) were used to characterize T. orientalis and to assess the infection intensity, respectively. Of 232 blood samples tested, T. orientalis DNA was detected in only 2.2% of samples using conventional PCR; two genotypes buffeli (1.3%; 3/232) and type 5 (0.9%; 2/232) of T. orientalis were detected. Phylogenetic analysis revealed that the buffeli MPSP sequences from Ethiopia were closely related to those reported from Kenya, Sri Lanka and Myanmar, and type 5 sequences from Ethiopia grouped with those from Korea, Japan, Vietnam and Thailand. A higher number of samples (3.9%; 9/232) were test-positive by MT-PCR and four genotypes (buffeli, chitose, ikeda and type 5) of T. orientalis were detected. The average intensity of infections with genotypes buffeli (DNA copy numbers 11,056) and type 5 (7508) were significantly higher (P<0.0001) than the pathogenic genotype ikeda (61 DNA copies). This first insight into T. orientalis from cattle in Ethiopia using MPSP gene provides a basis for future studies of T. orientalis in various agroclimatic zones and of the impact of oriental theilerosis on cattle in this and other countries of Africa.

Investigating the first outbreak of oriental theileriosis in cattle in South Australia using multiplexed tandem PCR (MT-PCR).

This study investigated the first outbreak of oriental theileriosis in a herd of beef cattle in South Australia using a newly established multiplexed tandem PCR (MT-PCR) to identify, differentiate and quantitate the four genotypes (buffeli, chitose, ikeda and type 5) of Theileria orientalis recognised to occur in Australasia. Following clinical diagnosis of oriental theileriosis (based on clinical signs, laboratory findings and post mortem examination), 155 blood samples were collected from individual cows (n = 85) and calves (n = 70), and tested by MT-PCR. In total, 117 (75.48%) cattle were shown to be test-positive for T. orientalis. All four genotypes were detected, and ikeda had the highest prevalence (90.6%; 106/117), followed by buffeli (83.8%; 98/117), chitose (18.8%; 22/117) and type 5 (5.1%; 6/117). Mixed infections with genotypes buffeli and ikeda had a higher prevalence (55.5%; 65/117) than any other combination of genotypes. The prevalences of buffeli and ikeda were significantly higher (P<0.005) than those of chitose and type 5. The average intensity of infection with genotype ikeda (329,775 DNA copies) was significantly higher (P<0.0001) than buffeli (212,843) and chitose (125,462). This study reinforces the utility of MT-PCR as a diagnostic tool for rapidly investigating oriental theileriosis outbreaks in cattle herds and as a pre-movement screening test for preventing the introduction of this disease into non-endemic regions.

Theileria infection in domestic ruminants in northern Ethiopia.

Piroplasmosis caused by different tick-borne hemoprotozoan parasites of the genera Theileria and Babesia is among the most economically important infections of domestic ruminants in sub-Saharan Africa. A survey for piroplasm infection was conducted in three locations in Northern Ethiopia. Of 525 domestic ruminants surveyed, 80% of the cattle, 94% of the sheep and 2% of the goats were positive for different Theileria spp. based on PCR of blood followed by DNA sequencing. Sheep had a significantly higher rate of infection compared with cattle (P<0.0003) and both sheep and cattle had higher rates of infection compared to goats (P<0.0001). Four species of Theileria were detected in cattle: T. velifera, T. mutans, T. orientalis complex and T. annulata with infection rates of 66, 8, 4, and 2%, respectively. This is the first report of T. annulata, the cause of Tropical Theileriosis in Ethiopia. Of the two Theileria spp. detected in small ruminants, T. ovis was highly prevalent (92%) in sheep and rare in goats (1.5%) whereas T. seperata was infrequent in sheep (2%) and rare in goats (0.4%). None of the animals were positive for Babesia spp.; however, Sarcocystis capracanis and S. tenella were detected in one goat and a sheep, respectively. The widespread distribution of Theileria spp. among cattle in northern Ethiopia including the virulent T. annulata and more mildly pathogenic T. mutans and T. orientalis, and the high infection rate in sheep with the usually sub-clinical T. ovis indicate extensive exposure to ticks and transmission of piroplasms with an important economic impact.