PCR-based detection of Theileria ovis in Rhipicephalus bursa adult ticks

Tick-borne diseases in ruminants are common in tropical and subtropical regions and lead to meat and milk production losses. In this study, polymerase chain reaction (PCR) was used to assess the presence of Theileria ovis in Rhipicephalus bursa ticks. We have demonstrated that the PCR enabled detection of T. ovis in field isolates of R. bursa collected from naturally infested sheep and goats in eastern Turkey. The sampling was done in spring season (between May and June 2004). A total of 420 R. bursa were collected and randomly selected 192 number of them (97 female and 95 male) were dissected. Primers specific for 520 bp fragments small subunit ribosomal RNA (ssu rRNA) gene of T. ovis amplified products from 37 of the 192 (19.27%) samples. The parasite was detected in 17 (17.52%) female and in 20 (21.05%) male ticks. Two T. ovis amplicons from the tick samples were purified and sequenced. The resulting sequences were identical to the nucleotide sequence of the Turkish sheep strain of T. ovis. These results showed that R. bursa might play an important role in the field as a natural vector of T. ovis.     

Clinical features associated with seroconversion to Anaplasma marginale, Babesia bigemina and Theileria parva infections in African cattle under natural tick challenge

A longitudinal study was conducted in Southeast Uganda for 14 months on 640 Zebu cattle kept under natural tick challenge, with a view to identifying clinical features for prediction of seroconversion to Anaplasma marginale, Babesia bigemina and Theileria parva infections. Physical examination, condition scoring and tick counts were undertaken on all cattle every 4 weeks. In addition, 5300 sera were collected and analysed for antibodies against A. marginale, B. bigemina and T. parva infections using the enzyme-linked immunosorbent assay (ELISA). The major clinical features compiled included weight loss, fever (rectal temperature), anaemia (packed cell volume), pallor of mucous membranes, lymph node enlargement, staring coat, diarrhoea and lacrymation. The risk factors included tick challenge at village level, sex, age, Rhipicephalus spp. density and Boophilus spp. density on individual animals. Using a binary logistic regression model, the clinical features and risk factors were analysed. The results suggest that increasing rectal temperature was associated with increased probability for seroconversion to A. marginale, while high level of Rhipicephalus spp. density and increasing packed cell volume (PCV) were significantly associated with reduced probability of seroconversion. Although statistically significant, none of the factors had large effects, with odds ratios (OR) of 0.87, 1.15 and 0.98 for Rhipicephalus spp. density, rectal temperature and PCV, respectively. For B. bigemina infection, a high level of Boophilus spp. density, anaemia and staring coat were significantly associated with increased probability of seroconversion (OR 1.50, 1.78, 1.37, respectively). Presence of lacrymation and old age were associated with reduced probability of seroconversion (OR 0.52, 0.86 respectively). For T. parva infection, lymph node enlargement (OR 1.30) was associated with increased probability of seroconversion, while high Rhipicephalus spp. density and increasing packed cell volume (PCV) were associated with reduced probability of seroconversion (OR 0.68 and 0.98, respectively). In conclusion, presence and intensity of the respective tick vectors for tick-borne diseases, age and clinical features such as anaemia, fever, staring coat, lymph node enlargement and lacrymation are indicators for seroconversion to A. marginale, B. bigemina and T. parva infections in cattle. These indicators for seroconversion could be exploited in the development of decision support tools for clinical diagnosis of tick-borne diseases.     

Identification of Rickettsia felis in fleas but not ticks on stray cats and dogs and the evidence of Rickettsia rhipicephali only in adult stage of Rhipicephalus sanguineus and Rhipicephalus haemaphysaloides

Rickettsia spp. are zoonotic pathogens and mainly transmitted by various arthropod vectors, such as fleas, ticks, and lice. Previous epidemiological studies indicated that ectoparasites infested on dogs or cats may be infected by Rickettsia spp., and transmit them to human beings accidentally. In this study, the prevalence of Rickettsia infection was evaluated using fleas and ticks from stray dogs and cats in Taiwan. A total of 158 pools made by 451 cat fleas (Ctenocephalides felis) from 37 dogs and 4 cats were used for analysis. Besides, 386 Rhipicephalus ticks collected from the other 62 stray dogs were included in this study. Nymphal and adult ticks were individually analyzed but larvae were separated into 21 pools for molecular detection. Partial sequencing analysis of the gltA gene was applied for Rickettsia identification. The results showed that 44.3% (70/158) of the cat flea pools were harboring Rickettsia DNA. Although 6.9% (13/187) of adult ticks were infected with Rickettsia, neither larval pools nor nymphal ticks were found to contain Rickettsia DNA. According to the results of sequencing analyses, all Rickettsia PCR-positive cat flea pools were infected with R. felis, and all Rickettsia PCR-positive adult ticks were infected with R. rhipicephali. The results of this study demonstrated that C. felis but not Rhipicephlus sanguineus (the brown dog tick) and Rh. haemaphysaloides collected from stray animals in Taiwan could be infected the zoonotic pathogen R. felis. Moreover, R. rhipicephali was only identified in adult stage of Rhipicephalus sanguineus and Rh. haemaphysaloides.     

Concomitant infection of cattle with the vaccine strain Anaplasma marginale ss centrale and field strains of A. marginale

Bovine anaplasmosis, caused by Anaplasma marginale, the intraerythrocytic rickettsia, is controlled by vaccination with live Anaplasma marginale ss centrale (A. centrale), a subspecies of relatively low pathogenicity. We have experimentally demonstrated that an animal primarily infected with A. marginale, or with the related vaccine subspecies A. centrale can be infected with the heterologous subspecies, and carries both bacteria. The co-infection was detected in experimentally cross-infected calves for up to 3 months after the last inoculation with the heterologous subspecies. The occurrence of characteristic cyclic rickettsemia of A. centrale and A. marginale was observed by examination of Giemsa-stained blood smears, or by the presence of specific rickettsial DNA confirmed in PCR assays based on specific msp1a and msp4 for A. marginale, and on specifically designed msp3 and msp4 primers for A. centrale. Sequence analysis of msp4-specific fragments for each subspecies revealed the presence of dual infection in both calves on days 30 and 60 after cross-inoculation with the heterologous Anaplasma subspecies. The experimental cross-infection of calves clearly demonstrated that the concept of "infection exclusion" does not apply to Anaplasma infection in cattle; as there was no infection exclusion of A. marginale in A. centrale-infected cattle, and vice versa. The present results confirmed our previous findings that cattle grazing in an anaplasmosis-endemic field were subject to concomitant infection with both the vaccine A. centrale and the field A. marginale strains.     

Erythrocytic oxidative damage in crossbred cattle naturally infected with Babesia bigemina

This study aimed to determine the erythrocytic lipid peroxidation and haemoglobin oxidation as contributory factors causing anaemia in cattle (Friesian × Egyptian native breed) infected with Babesia bigemina. Blood was collected from 32 cows infected with B. bigemina along with 18 healthy cows as controls for determination of erythrocytic malondialdehyde (MDA), blood methaemoglobin (MetHb), plasma free haemoglobin (PHb), corpuscular osmotic fragility (COF), red blood cell count (RBC), total haemoglobin (Hb) and packed cell volume (PCV). Percentage of parasitaemia varied from 14% to 36%. MDA, MetHb, COF and PHb were significantly increased (P < 0.001) in infected cows versus controls. Parasitaemia was positively correlated (P < 0.001) with MDA, MetHb, COF and PHb. MDA was positively correlated (P < 0.001) with COF and PHb and negatively correlated (P < 0.001) with RBC, Hb and PCV. MetHb was negatively correlated (P < 0.001) with RBC, Hb and PCV and positively correlated (P < 0.001) with COF. In conclusion, B. bigemina infection in cattle is associated with a parasitic burden-dependent corpuscular oxidative damage as indicated by membrane lipid peroxidation and methaemoglobin formation, which are contributed to COF and intravascular haemolysis.     

Serological Survey of Babesia bovis and Anaplasma marginale in cattle in Tete Province,Mozambique

A serological survey of bovine babesiosis and anaplasmosis in communal cattle was conducted in the northwestern province of Tete, Mozambique. Blood was collected from cattle ranging from 4 to 15 months old from randomly selected farms from six districts. Thirty-nine per cent of all 478 calves tested in Tete Province were seropositive to the ELISA for Babesia bovis antibodies and 63% of all calves were seropositive in the card agglutination test for Anaplasma marginale. Seroprevalence of B. bovis ranged from 22.8% in Tete City District to 48.1% in Angonia District. For A. marginale, it ranged from 34.4% in Angonia District to 87.3% in Moatize District. The dominant factor affecting seroprevalence for both haemoparasites was district and there was a trend for higher intensity of tick control to be associated with a higher seroprevalence of B. bovis and a lower seroprevalence of A. marginale. The obvious differences were the low prevalence of B. bovis in Tete City Council District and the low prevalence of A. marginale in Angonia District. The levels of exposure to B. bovis seen in our study are well below any that could be considered to be consistent with endemic stability, yet they are sufficiently high to ensure that clinical disease would be a risk. The seroprevalence of A. marginale, however, suggests that endemic stability with respect to this disease could exist in districts other than Angonia. There was no strong and consistent relationship between the intensity of control and the likelihood of seropositivity to either of the diseases.     

Transmission of Theileria parva in the Traditional Farming Sector in the Southern Province of Zambia during 1997–1998

The incidence of first contact with the protozoan Theileria parva was determined in two traditional cattle herds in the Southern Province of Zambia during a period of average rainfall in 1997 and 1998, following a drought in the previous two years. Compared to that period, there was a marked increase in the number of rainy season first contacts attributable to transmission by Rhipicephalus appendiculatus adults. However, there were still more dry season contacts that resulted from nymphal transmission. These results highlight the important role that climate plays in the transmission of theileriosis in the Southern Province of Zambia.     

Transmission of Theileriosis in the Traditional Farming Sector in the Southern Province of Zambia during 1995–1996

The incidence of first contact with the protozoan Theileria parva was determined in three traditional cattle herds in the Southern Province of Zambia in 1995 and 1996. The majority of first contacts occurred during the dry season in June, July and August, at a time of nymphal activity and in the absence of Rhipicephalus appendiculatus adults, indicating that larva to nymph transmission plays a more prominent role than nymph to adult transmission under the prevailing conditions.     

Orientation of bovine CTL responses towards PIM, an antibody-inducing surface molecule of Theileria parva, by DNA subunit immunization

East Coast fever, an acute lymphoproliferative disease of cattle, is caused by the apicomplexan parasite Theileria parva. Protective immunity is mediated by CD8(+) cytotoxic T lymphocytes directed against schizont-infected cells. The polymorphic immunodominant molecule, although an antibody-inducing surface molecule of the schizont, has been hypothesized to play a role in protective immunity. In order to evaluate the immunogenicity of PIM for inducing CTL, cattle were immunized with PIM in isolation from other T. parva antigens, forcing the presentation of PIM-derived epitopes on the MHC class I molecules. Although parasite-specific cytotoxicity was induced in both vaccinated animals, their immune response was clearly different. One animal generated MHC-restricted parasite-specific CTL against PIM while the other calf exhibited a strong PIM-specific proliferative response but non-MHC-restricted parasite-specific cytotoxicity. Only calf 1 survived a lethal sporozoite challenge. This DNA immunization technique with an antigen in isolation of CTL-immunodominant antigens might open possibilities for directing CTL responses against predefined antigens, such as strain cross-reacting CTL antigens.     

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 κ- 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.     

Characterization of pathogen-specific expression of host immune response genes in Anaplasma and Mycobacterium species infected ruminants

Anaplasma and Mycobacterium species are among the most prevalent bacterial pathogens in European red deer (Cervus elaphus) in south-central Spain and are known to modify gene expression in ruminants. In this study, we used microarray hybridization and real-time RT-PCR analyses to characterize global gene expression profiles in red deer in response to Anaplasma ovis and A. ovis/Mycobacterium bovis/Mycobacterium avium sub. paratuberculosis (MAP) infections, compare the expression of immune response genes between red deer infected with A. ovis, M. bovis and A. ovis/M. bovis/MAP, and characterize the differential expression of immune response genes identified in red deer in cattle infected with M. bovis and Anaplasma marginale. Global gene differential expression in A. ovis- and A. ovis/M. bovis/MAP-infected deer resulted in the modification of common and pathogen-specific cellular biological processes. The differential expression of host immune response genes showed pathogen and host-specific signatures and the effect of infection with multiple pathogens on deer immune response. These results suggested that intracellular bacteria from Anaplasma and Mycobacterium genera produce similar genes expression patterns in infected ruminants. However, pathogen and host-specific differences could contribute to disease diagnosis and treatment in ruminants.     

Detection of Theileria and Babesia species in ticks collected from cattle

The present study was carried out to detect tick species that infest cattle, and Theileria and Babesia species transmitted by these ticks in Kayseri province (Turkey). A total of 300 cattle were examined for tick infestations. Of the 300 cattle, 117 (39%) were infested with ticks. A total of 1160 ticks belonging to 11 Ixodid genera were collected from the infested animals and their shelters. The most prevalent tick species was Boophilus annulatus 26.37% (306/1160) followed by Hyalomma marginatum marginatum 21.12% (245/1160) and Rhipicephalus turanicus 18.7% (217/1160). The collected ticks were separated into 43 tick pools, according to their species. These pools were examined for bovine Theileria and Babesia species (Theileria sp., Babesia sp., Theileria annulata, T. buffeli/orientalis, Babesia bigemina, B. bovis and B. divergens) by using the reverse line blotting method (RLB). Of the 43 tick pools examined, 6 (14%) were infected with B. bigemina, 4 (9.3%) with T. annulata, and 1 (2.3%) with Babesia sp., whereas 1 (2.3%) displayed mixed infection with T. annulata + B. bigemina. The sequence and phylogenetic analyses of Babesia sp., which could not be identified to the species level by RLB, were performed. In the phylogenetic tree, Babesia sp. (Kayseri 1) grouped with Babesia sp. (Kashi 2), Babesia sp. (Kashi 1), Babesia sp. (Xinjiang) and B. orientalis with 96.8-100% identity.     

Immune response of BALB/c mouse immunized with recombinant MSPs proteins of Anaplasma marginale binding to immunostimulant complex (ISCOM)

Anaplasmosis, caused by Anaplasma marginale, results in significant economic losses of cattle in tropical and subtropical regions worldwide. Six major surface proteins (MSPs) were well characterized and designated as MSP1, MSP2, MSP3, MSP4, and MSP5. The objective of this study was to evaluate the humoral immune response of BALB/c mice against the recombinant MSPs, incorporated into immunostimulating complex (ISCOM). The recombinant proteins purified by Ni-NTA columns were incorporated into ISCOM and ISCOMATRIX by the lipid film hydration method. BALB/c mice immunized with ISCOM/rMSPs and ISCOMATRIX/rMSPs vaccines produced whole IgG, IgG1, and IgG2a, in contrast to the negative groups (PBS and ISCOMATRIX adjuvant). All groups that received antigen responded specifically against the rMSPs by Western blotting, showing the rMSP1a (60-105kDa), rMSP1b (100kDa), rMSP4 (47kDa), and rMSP5 (29kDa). Additional studies will have to be performed in cattle to evaluate the humoral and cellular mechanisms of this subunit vaccine and their possible use as protective vaccines against homologous and heterologous strains of A. marginale.     

Clinical and Laboratory Findings in Equine Piroplasmosis

The objective of this study was to evaluate equine piroplasmosis (EP) as a cause of morbidity in horses in Sardinia (Italy), describe the clinical signs and altered hematologic and biochemical parameters, and illustrate response to different treatments. Among 44 horses suspected of tick-borne disease, 38 were polymerase chain reaction (PCR) positive for Theileria equi (n = 27) or Babesia caballi (n = 6), whereas five were positive for both protozoans. Typical clinical features of piroplasmosis were seen in some of the horses, whereas others had nonspecific mild symptoms. Hematologic findings revealed involvement of the three blood cell lineages (anemia, leukopenia or leukocytosis, thrombocytopenia), and biochemical variations were related to increased bilirubin, alteration of serum phosphorus, and hypoalbuminemia. We suggest that the two protozoans are the most important causative agents of equine tick-borne disease in this geographic area, and we observe that different clinical features are associated with the disease; in addition to the typical aspects of piroplasmosis, characterized by fever, pale mucous membranes, and icterus, we can signal other nonspecific mild signs such as weight loss, weight loss associated with an insignificant leukopenia, or weight loss associated with depression, anorexia, and mild hyperbilirubin. The study is intended as a practical contribution for veterinary practitioners because it describes different clinical presentations and laboratory findings of EP, suggests diagnostic and therapeutic approaches to the disease, and shows diffusion of the disease in a Mediterranean region.     

Slide Enzyme-Linked Immunosorbent Assay for the Diagnosis of Babesia bigemina Infection in Bovines

A slide enzyme-linked immunosorbent assay (SELISA) for the diagnosis of Babesia bigemina infection in cattle was standardized. Acetone-fixed whole Babesia bigemina-infected erythrocytes on micro-slides were immunoreacted with bovine serum samples followed by antibovine horseradish peroxidase conjugate and developed using diaminobenzidine tetrahydrochloride as a substrate. The positive immunoreactivity (staining pattern) was visualized in the form of dark brown piroplasms. Using the laboratory-standardized SELISA with a sensitivity of 94.4%, the seroprevalence of babesiosis was studied in cattle from two endemic areas of the disease. In comparison to IFAT, SELISA detected higher number of serum samples positive for bovine babesiosis.     

Mortality and commercial off-take rates in adult traditional cattle of Zambia

A cohort study was conducted in the livestock-wildlife interface areas of Zambia to determine cattle mortality and commercial off-take rates among adult cattle as well as factors influencing them. A total of 416 animals from 43 herds were followed up for one year and animals were individually identified and their fate was indicated as sold, slaughtered, dead or present as appropriate. The overall mortality incidence risk was estimated at 7.5%. Cattle in Kazungula were at a greater risk of dying compared to those in Blue Lagoon and Lochnivar. Annual off-take was estimated at 13.7% (8.1–19.3%), unadjusted values, and 16.4% (8.1–24.5%) after adjusting for sampling fraction in primary sampling units (herds) and area stratification. Area variations were observed with Kazungula recording the highest in both instances, which was attributed to a contagious bovine pleural pneumonia (CBPP) outbreak. Herd size and gender were observed to influence cattle mortality rates. Cattle in the middle-sized herds (50–150 cattle) recorded high mortality rates (OR = 3.91) compared to smaller herds (10–50) and so were females compared to males (OR = 4.16). The logistic regression model showed that cattle death was influenced by managerial factors and that off-take rates tend to increase in the face of disease outbreaks.     

Enzymes of glucose and glycerol catabolism in in vitro-propagated Theileria parva schizonts

Theileria parva schizonts propagated in vitro in peripheral blood lymphocytes were purified and assayed for key enzymes of glucose and glycerol catabolism and the citric acid cycle. The activities of glycolytic enzymes were in the range of 21-100 nmol/min/mg protein. Glycerol kinase and alpha -glycerophosphate dehydrogenase activities were more than 16 times lower than the activities of other enzymes catalysing the oxidation of the triose phosphates to lactate. It was suggested that the catabolism of glycerol is negligible and that glucose is catabolized to lactate via the Embden-Meyerhof pathway. The activities of the enzymes catalysing the section of the citric acid cycle that involves the formation of citrate to succinyl-CoA were consistently very low (less than 2.0 nmol/min/mg protein), indicating that this part of the cycle plays a minor role in this parasite. Enzyme activities of the cycle catalysing the formation of succinate from oxaloacetate were relatively higher than those catalysing other sections of the citric acid cycle, suggesting that this section of the cycle could be important to the parasite. Pyruvate carboxylase activity was more than 10 times that of phosphoenolpyruvate carboxykinase. It was suggested that pyruvate could be carboxylated to oxaloacetate. Taken together, these results suggest that the catabolism of glucose in Theileria parva schizonts is mainly via the Embden-Meyerhof pathway and that the citric acid cycle plays a minor role in energy production.     

Seroprevalence Estimation and Risk Factors for <Emphasis Type="Italic">A. marginale</Emphasis> on Smallholder Dairy Farms in Tanzania

A cross-sectional serological survey of A. marginale was conducted on 200 randomly selected smallholder farms in each of the Tanga and Iringa Regions of Tanzania between January and April 1999. Sera, from dairy cattle of all ages, sexes and breeds were tested for antibodies against A. marginale using an indirect enzyme-linked immunosorbent assay. Antibodies to A. marginale were present in cattle throughout the study areas and the overall prevalence was 20% for Tanga and 37% for Iringa. The forces of infection based on the age seroprevalence profile were estimated at 8 for Tanga and 15 for Iringa per 100 cattle years-risk, respectively. In both regions, seroprevalence increased with age (β = 0.01 and 0.017 per year of age, p < 0.005, in Tanga and Iringa, respectively). Older animals in Iringa were significantly and negatively associated with decreased seropositivity (β = −0.002, p = 0.0029). Further results of logistic regression models reveal that geographic location of animals in Tanga was associated with seropositivity (odds ratio (OR) = 2.94, p = 0.005, for Tanga Rural and OR = 2.38, p = 0.066, for Muheza). Animals acquired as a gift in Iringa had higher odds for seropositivity than brought-in cattle (OR = 2.44, p = 0.005). Our study has identified and quantified some key risk factors that can guide planners devising disease control strategies.     

Bartonella and Babesia infections in cattle and their ticks in Taiwan

Bartonella and Babesia infections and the association with cattle breed and age as well as tick species infesting selected cattle herds in Taiwan were investigated. Blood samples were collected from 518 dairy cows and 59 beef cattle on 14 farms and 415 ticks were collected from these animals or in a field. Bartonella and Babesia species were isolated and/or detected in the cattle blood samples and from a selected subset (n = 254) of the ticks either by culture or DNA extraction, PCR testing and DNA sequence analysis. Bartonella bovis was isolated from a dairy cow and was detected in 25 (42.4%) beef cattle and 40 (15.7%) tick DNA samples. This is the first isolation of B. bovis from cattle in Asia and detection of a wide variety of Bartonella species in Rhipicephalus microplus. Babesia spp. were detected only on one farm from dairy cows either infected by Babesia bovis (n = 10, 1.9%) or B. bigemina (n = 3, 0.6%).     

The Effect of Management Factors on the Seroprevalence of Anaplasma marginale in Bos indicus cattle in the Mexican Tropics

A cross-sectional study with a two-stage design and proportional distribution was carried out to determine the effect of management factors on the seroprevalence of Anaplasma marginale in Bos indicus cattle in the Mexican tropics. Serum was obtained from 384 cattle aged 1-2 years on 92 farms. The number of samples was proportionally distributed according to the number of farms in eastern Yucatan. Antibody activity against A. marginale was assessed using the card agglutination test. A primary screening using a 2 x K contingency table of the exposed variables with the outcomes was performed. All variables for which p < 0.20 were included in a fixed-effects log regression. The seroprevalence in the cattle was 69.75% (SE +/- 0.02). Sixty-four per cent of the farms had a seroprevalence > or = 75%. The risks related to managemental factors were stocking density ( > or = 1 animal/ha, OR = 10.94), type of acaricide (pyrethroids, OR = 3.8), dipping interval ( > 60 days, OR = 0.13) and type of veterinary instruments used (needles, scalpels, ear tattoos, and dehorners, OR = 0.17).