Molecular prevalence of Babesia bigemina and Trypanosoma evansi in dairy animals from Punjab, India, by duplex PCR: a step forward to the detection and management of concurrent latent infections.

Specific duplex polymerase chain reaction (PCR) was employed on 411 (386 cattle and 25 buffaloes) blood samples of dairy animals from 9 districts of Punjab, India, for simultaneous detection of Babesia bigemina and Trypanosoma evansi. The results were compared and correlated with conventional Giemsa stained thin blood smear (GSTBS) examination and haematological alterations to know the clinical status and pathogenicity of infections. The Bg3/Bg4 and TR3/TR4 primers were used in duplex PCR for B. bigemina and T. evansi amplified products of 689 bp and 257 bp, respectively. The overall prevalence by duplex PCR was found to be 36.49, 2.43, and 3.41% for T. evansi, B. bigemina, and dual infection, respectively. A more significant difference was observed for dual infection status (P ≤ 0.005) as compared to T. evansi (P ≤ 0.05) and B. bigemina (P ≤ 0.01) among various districts under study. A very low prevalence of T. evansi (0.73%) and B. bigemina (0.48%) was seen by GSTBS. The highly sensitive, specific, and cost-effective duplex PCR was able to detect latent T. evansi and B. bigemina infection in cattle and buffaloes. Haematological evaluation revealed marked pathology in B. bigemina infected group and in dual infected group in contrast to that infected with T. evansi alone.

Virulence attenuation of Babesia gibsoni by serial passages in vitro and assessment of the protection provided by the immunization against the passaged isolate in dogs.

The virulence of the Babesia gibsoni Oita isolate was attenuated by serial passages in vitro by using the microaerophilus stationary phase (MASP) technique. After 400 serial passages, the virulence of the isolate was found to be attenuated. This was evidenced by the response of two dogs inoculated intravenously with 10(9)B. gibsoni passaged isolate. Specific antibodies were produced at a titer of 1:20,480, as detected by the fluorescent antibody test (IFAT). These results suggested that the serial passages of B. gibsoni reduced its virulence while retaining its antigenicity. The dogs that were inoculated with the attenuated isolate (1 and 2) and two naïve dogs (3 and 4) were challenged by intravenous inoculation of 2×10(8) infected erythrocytes of the virulent Oita isolate. Protection afforded by exposure to the attenuated isolate was evidenced by a lower parasitemia in dogs 1 and 2 with a rapid decrease to nondetectable levels, accompanied by a slight decrease in the PCV that returned to normal values. Dogs 3 and 4 developed typical acute clinical signs, including severe anemia and hyperthermia. These results suggested that the attenuated isolate was a candidate for live vaccine.

Assessment of exposure to piroplasms in sheep grazing in communal mountain pastures by using a multiplex DNA bead-based suspension array.

BACKGROUND: Piroplasms are tick-borne hemoprotozoans with a major impact on extensive management systems. Detection of sub-clinical low-level carriers, which can act as source of infection for vector ticks, is key to protect livestock trade and facilitate preventive control programs. The purpose of this study was to develop a method for the detection of ovine piroplasms and to use it in a field study aimed at investigating piroplasms infection in semi-extensive production systems in the Basque Country (northern Spain). METHODS: A DNA bead-based suspension array using the Luminex xMAP technology that included a generic Theileria-Babesia control probe, 6 species-specific probes, and an internal control probe was developed to detect and identify piroplasms that infect sheep. To monitor piroplasm infection in clinically healthy sheep from 4 flocks that share communal mountain pastures, blood samples were collected during 2 grazing seasons. RESULTS: Piroplasms were detected in 48% (214/446) of blood samples, nearly half of them (49.1%, 105/214) as mixed infections. Five different piroplasms were identified: Theileria sp. OT3 in 34.8% of the samples, Theileria ovis in 20.9%, and at lower prevalences Babesia motasi (12.3%), Theileria luwenshuni/OT1 (10.5%) and Babesia ovis (6.3%). Despite differences among flocks associated to differences in management, an increasing trend in the incidence of piroplasm infection with increasing age of animals after increased tick exposure was observed. This increment could be attributed to continued re-infection associated with re-exposure to ticks at grazing. Ticks were collected from animals (4 species) and vegetation (8 species), and associations between tick abundance seasonality and risk of infection with the different piroplasms were established. CONCLUSION: The multiplex Luminex xMAP procedure is a rapid and high throughput technique that provided highly specific and sensitive identification of single and mixed piroplasm infections in blood of sheep carriers. This study confirmed a situation of endemic stability for piroplasm infection in the region, where infection is present in the absence of clinical signs, and mountain grazing allows for sufficient inoculation rates to maintain such situation.

Assessment of renal dysfunction using urinary markers in canine babesiosis caused by Babesia rossi.

Renal damage is deemed a common, yet poorly documented, complication in canine babesiosis. Serum urea and creatinine are insensitive and non-specific markers of early renal dysfunction and their measurements are influenced by hemolysis caused by babesiosis. Therefore, the aim of this study was to use urinary markers to assess the localization and degree of renal dysfunction in dogs with Babesia rossi infection. Urinary immunoglobulin G (uIgG) and urinary C-reactive protein (uCRP) were measured as markers for glomerular dysfunction, while urinary retinol-binding protein (uRBP) was used as a marker for tubular dysfunction. Eighteen dogs presenting with uncomplicated babesiosis were included and compared with eight clinically healthy dogs. Previously validated commercial ELISA kits were used for the measurement of uIgG, uCRP, and uRBP. Results were related to urinary creatinine concentrations (c). Dogs with babesiosis had significantly higher concentrations of all three measured urinary markers compared to healthy dogs. Except for urinary protein/c ratio (UPC), routine urinary and serum markers for renal function (urine specific gravity (USG), serum urea and creatinine (sCr)) were not significantly different between dogs with babesiosis and healthy dogs. All three urinary markers were positively correlated with each other and with UPC. The data supports the presence of both glomerular and tubular dysfunction in dogs suffering from uncomplicated B. rossi infection. Urinary markers were superior to USG, serum urea and creatinine concentrations for the early detection of renal dysfunction in dogs with babesiosis.

Prevalence of livestock diseases and their impact on livelihoods in Central Equatoria State, Southern Sudan.

A participatory epidemiological (PE) study was conducted in Kajo Keji and Yei Counties, Central Equatoria State, southern Sudan to assess the impact of livestock diseases on livelihoods. A serological survey of tick-borne diseases was conducted to supplement the PE study. PE data collection tools consisted primarily of focus group interviews and key informant interviews supplemented by observation. Information was collected on the social context, history and species of livestock kept. Constraints in livestock keeping were explored through description and probing. Proportional piling on the importance of different diseases and relative incidence scoring were also conducted. 243 sera were collected from cattle and tested for antibodies to Anaplasma marginale, Babesia bigemina, B. bovis, Theileria mutans and T. parva by ELISA. Additionally, 173 blood samples were collected for a PCR assay of T. parva. Livestock diseases were ranked as the most important constraint to livestock keeping. While East Coast fever was ranked as the most important disease in Kajo Keji, diarrhoea in small ruminants was reported as the most important disease in Yei. Serological analyses of the sera indicated that A. marginale, B. bigemina, T. mutans and T. parva were most prevalent. Prevalence of B. bovis was found to be low (4.0% and 7.4% in Kajo Keji and Yei, respectively). 35% of the samples screened with the T. parva p104 gene nested PCR assay were positive. The study concludes that while ECF is the most important disease in Kajo Keji, it was not the case in Yei. Additional epidemiological studies are proposed before control strategies are recommended.

Comparison of indirect fluorescent antibody test (IFAT) and slide enzyme linked immunosorbent assay (SELISA) for diagnosis of Babesia bigemina infection in bovines.

An indirect fluorescent antibody test (IFAT) and slide enzyme linked immunosorbent assay (SELISA) were standardized for the detection of antibodies specific to Babesia bigemina in experimentally infected bovine calves and subsequently used for the screening of naturally infected bovine and bubaline sera. In experimentally infected calves positive reactivity was detected in sera at the earliest on day 7 by both the tests. Serological studies for detection of B. bigemina specific antibodies in 180 cow and 120 buffalo serum samples procured from endemic zones of Uttar Pradesh and Punjab revealed 56.11% and 23.33% seropositivity, respectively, both by SELISA and IFAT. Variation in the reactivity pattern between these tests was found to be non significant. The sensitivity of SELISA was determined to be 94.85% whereas the specificity was 90.85% in comparison to IFAT. The agreement between the two tests by kappa statistics at 95% confidence interval revealed kappa- value of 0.853 that depicts almost a perfect degree of agreement. The findings employing experimental as well as test sera from cattle and buffalo from some of the tick infested zones of India suggested that SELISA could be a useful tool for seroprevalence studies on babesiosis, as the test is less cost intensive with high levels of sensitivity and specificity.

Assessment of primers designed for the subspecies-specific discrimination among Babesia canis canis, Babesia canis vogeli and Babesia canis rossi by PCR assay.

Canine babesiosis is an infectious disease caused by either Babesia gibsoni or Babesia canis protozoans. The latter is also classified under three different phylogenetic groups, referred to as subspecies B. canis canis, B. canis vogeli and B. canis rossi. The objective of the present study was to validate and standardize a PCR assay to discriminate the organisms at the subspecies level. First, the reference sequences of the 18S rRNA, 5.8S rRNA and 28S rRNA genes, including the internal transcribed spacer 1 (ITS1) and 2 (ITS2) of the most common species and subspecies of the genus Babesia were retrieved from the GenBank database. Subspecies-specific primers (BAB3, BAB4 and BAB5) and one genus-specific primer were designed from the alignment of the sequences. The PCR assays were evaluated in three different combinations of primer pairs in order to assure complete specificity for each reaction. The results of the tests had demonstrated effectiveness of the novel primer pairs BAB1/BAB3, BAB1/BAB4 and BAB1/BAB5 for the amplification of the subspecies-specific target fragments of 746 bp (B. c. canis), 546 bp (B. c. vogeli) and 342 bp (B. c. rossi) by PCR. The original enzymatic amplification assays with novel primers reported in this paper were confirmed to be a reliable tool for the specific discrimination among B. canis subspecies by single-step PCR assays.

Estimation of the transmission dynamics of Theileria equi and Babesia caballi in horses.

For the evaluation of the epidemiology of Theileria equi and Babesia caballi in a herd of 510 horses in SW Mongolia, several mathematical models of the transmission dynamics were constructed. Because the field data contain information on the presence of the parasite (determined by PCR) and the presence of antibodies (determined by IFAT), the models cater for maternal protection with antibodies, susceptible animals, infected animals and animals which have eliminated the parasite and also allow for age-dependent infection in susceptible animals. Maximum likelihood estimation procedures were used to estimate the model parameters and a Monte Carlo approach was applied to select the best fitting model. Overall, the results are in line with previous experimental work, and add evidence that the epidemiology of T. equi differs from that of Babesia spp. The presented modelling approach provides a useful tool for the investigation of some vector-borne diseases and the applied model selection procedure avoids asymptotical assumptions that may not be adequate for the analysis of epidemiological field data.

Estimated direct economic costs associated with tick-borne diseases on cattle in Tanzania.

Tick-borne diseases, namely, anaplasmosis, babesiosis, cowdriosis and theileriosis, constrain cattle production and improvement in Tanzania, leading to considerable economic losses. A simple spreadsheet model was used to estimate the economic losses resulting from production losses, treatment and control costs associated with tick-borne diseases (TBD) in Tanzania. Model parameters included the national cattle population, reported TBD morbidity, fatality risk, and chemotherapy and control measures used. The total annual national loss due TBD was estimated to be 364 million USD, including an estimated mortality of 1.3 million cattle. Theileriosis accounted for 68% of the total loss, while anaplasmosis and babesiosis each accounted for 13% and cowdriosis accounted for 6% of the total loss. Costs associated with mortality, chemotherapy and acaricide application accounted for 49%, 21% and 14% of the total estimated annual TBD losses, respectively, infection and treatment method milk loss and weight loss accounted for 1%, 6% and 9% of the total annual loss, respectively. Despite the inadequacies of the data used, the results give evidence that tick-borne diseases inflict substantial economic losses on cattle production and resource use in Tanzania.

Prevalence of blood parasites in cattle from wilayates of Annaba and El Tarf east Algeria.

Between June and September 2002, a preliminary study was conducted to assess the prevalence of blood parasites of cattle in eastern Algeria. Fifty-four bovines of different genotypes were submitted to clinical examination. From each animal, blood smears were made and stained by Giemsa. Four species of parasites, namely Theileria annulata, T. orientalis, Babesia bovis and Anaplasma marginale were encountered. Fifty animals carried single or multiple infections with blood parasites and four were found negative. The rate of single infections (72.3%, n = 39) was almost three times higher than multiple infections (20.3%, n = 11). The high percentage of single infections was recorded with T. annulata (53.7%). However single infection with Anaplasma marginale (7.4 %), B. bovis (5.6%) and T orientalis (5.6%) were very low compared to T. annulata infection. The rates of mixed infection were as follows: T. annulata/A. marginale 9.3%, T. annulata/T. orientalis 5.6%, A. marginale/T. orientalis 3.7% and B. bovis/A. marginale 1.9%.

Present status of tick-borne diseases in Sudan.

Ticks and tick-borne diseases are widespread in the Sudan, cause substantial economic losses and constitute major obstacles to the development of animal wealth. Most important among these diseases are tropical theileriosis, malignant ovine theileriosis, cowdriosis, babesiosis, anaplasmosis and avian spirochaetosis. However, knowledge about ticks and tick-borne diseases is still fragmentary and far from complete. The large number of tick species, the multplicity of transmitted agents and the diverse ecoclimatic zones of the Sudan provide a unique opportunity to host diverse research activities that could benefit other regions in Africa.

Survey of canine babesiosis in South Africa.

A questionnaire, designed to obtain qualitative information on a number of variables concerning canine babesiosis (biliary fever) in South Africa, was sent to 510 veterinary practices in late 1993. Of the 157 practices that responded, all were presented with cases of babesiosis and most were situated in Gauteng, the Western Cape and KwaZulu-Natal. Apart from the Western Cape, a winter-rainfall region, the prevalence of babesiosis cases in dogs was highest in summer. Most of the respondent practices treated between 1,000 and 5,000 sick dogs that included 100 to 500 babesiosis cases each year. Respondents identified cerebral babesiosis, enterorrhagia, 'red' or haemoconcentrated babesiosis, acute renal failure and pulmonary babesiosis or 'shock lung', amongst others, as the most prevalent forms of complicated ('atypical') babesiosis. Diminazene, imidocarb and trypan blue were the most popular antibabesials. Trypan blue was most often used in shocked patients, whereas diminazene and imidocarb were preferred when there was a high parasitaemia in the absence of shock. At least 19 antibabesial treatment regimens were used in practices. These comprised the use of single doses of antibabesial drugs; split doses with repeat injections, and combined drug variations, some of which are undesirable due to possible sterilisation of Babesia infection or potential toxicity. Side-effects were most commonly associated with imidocarb use. Ninety-six percent of respondents used supportive treatment (e.g. corticosteroids, vitamins and 'liver support') in all cases of babesiosis. The use of blood transfusion as supportive treatment varied according to practice and severity of the case. Most practices never cross-matched blood to be transfused, and transfusion reactions were rare. Diminazene was most frequently incriminated in cases where drug 'resistance' or relapses occurred. Cerebral and 'red' cases resulted in high mortality. Treatment of babesiosis costs the dog-owning public in South Africa more than R20 million each year. Information on the distribution and possible complicating role of Ehrlichia canis was obtained. Development of a vaccine was the first research priority identified.