Differential Bos taurus cattle response to Babesia bovis infection

Bovine babesiosis is a tick-borne disease caused by Babesia spp. haemoprotozoans. The disease is of great importance at tick enzootic unstable areas and hampers cattle production in several developing countries. The available immunisation alternatives are pre-immunition and attenuated vaccines. Despite being efficient and protective, they are unsafe as they use cattle blood cells as inoculum and may potentially spread other diseases. Another alternative to help in babesiosis control would be the identification of genetically resistant cattle to Babesia bovis infection. The objective of this work was to phenotype cattle based on primary response against B. bovis infection. Two-hundred and forty half-sib Hereford and Aberdeen Angus heifers (120 animals from each breed), 12-18-month-old na?ve cattle, originated from a tick-free area in Southern Brazil, were used in the experiment. Animals were monitored following an inoculation with 1x10(7)B. bovis parasitised erythrocytes. Results showed three different phenotypes: 1-'susceptible', animals with babesiosis clinical signs that received treatment to avoid death; 2-'intermediate', animals with clinical signs: parasitaemia, >or=21.5% reduction in packed cell volume (PCV) and increase in body temperature when compared to their pre-challenge physiological parameters, no specific treatment was needed as animals self recovered from the disease, and 3-'resistant', animals without clinical signs that showed B. bovis presence in blood smears, <21.5% PCV reductions, with little or no increase in body temperature and no need for babesiosis treatment. The frequencies of each phenotype were: 45.4, 26.7, and 27.9%, respectively, demonstrating the existence of phenotypic variation for B. bovis in Bos taurus cattle.     

Bovine NK cells acquire cytotoxic activity and produce IFN-gamma after stimulation by Mycobacterium bovis BCG- or Babesia bovis-exposed splenic dendritic cells

Early interactions of innate immune cell populations, such as dendritic cells (DC) and natural killer (NK) cells, can affect the ability of the acquired immune response to control infection of intracellular microorganisms. In this study, we investigated the activation of bovine NK cells by CD13(+) splenic DC stimulated with either Mycobacterium bovis BCG or Babesia bovis merozoites. Splenic DC were used either immediately after selection (cytokine(-)) or after exposure to GM-CSF, IL-4 and Flt3L for 72 h (cytokine(+)). Phenotypic analyses showed up-regulation of MHCII, CD80 and CD86 on cytokine(+) DC when compared to cytokine(-) DC. Purified NK cells (CD335(+)CD3(-)CD2(+/-)CD8alpha(+/-)) were co-cultured with microbial-exposed cytokine(-) DC or cytokine(+) DC in either transwell or cell-to-cell format and NK cell IFN-gamma production and cytotoxicity were assessed. NK cell IFN-gamma production was dependent on cell-to-cell contact. Microbial-stimulated cytokine(+) DC induced significantly more IFN-gamma production from NK cells than cytokine(-) cells. In contrast, cytotoxicity and perforin up-regulation were more pronounced in NK cells cultured with cytokine(-) DC than cytokine(+) DC. Therefore, activation of bovine NK cells by microbial-stimulated CD13(+) splenic DC is influenced by the maturation state of the DC suggesting different roles for the splenic DC during disease-induced maturation.     

The transmission of Mycobacterium bovis infection to cattle

The prevalence of Mycobacterium bovis infection in cattle is increasing rapidly in some countries, including the UK and Ireland. The organism infects a wide range of mammalian hosts, and eradication of the disease is difficult if there is an extensive reservoir in the wildlife population. Existing evidence suggests that wildlife vectors include the European badger in the UK and Ireland, the brush-tailed possum and ferret in New Zealand and ungulates in some other countries. Cattle grazing field boundaries or short swards are at particularly high risk, since the chance of contact with the intermediate host or their excreta is increased. There is evidence that the transmission of the disease between cattle following movement accounts for 10-15% of outbreaks in the British Isles and that transmission can occur across farm boundaries. The prevalence the prevalence of single reactors in herds suggested that within-herd transmission was not common. In herds with infected cattle, spreading slurry is a risk factor, which can be minimised by prolonged storage of the slurry, by spreading it on fields not used for grazing or by soil injection. M. bovis also survives in water and may enter the respiratory tract during drinking. It is concluded that M. bovis infection in cattle can be transmitted by a number of routes, some of which can be controlled by appropriate husbandry, but that circumstantial evidence suggests that the existence of a widespread intermediate host is the greatest contributor to infection in cattle.     

Molecular and serological prevalence of Babesia bovis and Babesia bigemina in cattle from central region of Syria

A total of 207 bovine blood samples were collected from clinically healthy cattle bred in central region of Syria and examined by Giemsa-stained blood smears, nested PCR, ELISA, and IFAT to determine the molecular and serological prevalence of Babesia bovis and B. bigemina. All samples were negative to Babesia spp. by microscopic examination of blood smears. On the other hand, the overall prevalence of B. bovis and B. bigemina was 9.18% and 15.46% by nPCR, 15.46% and 18.84% by ELISA, and 18.36% and 21.74% by IFAT, respectively. Mixed infections were detected in a total of 5 samples (2.4%) by nPCR, 16 (7.73%) by ELISA and 27 (13.04%) by IFAT. Statistically significant differences in the prevalence of the two infections were observed on the basis of age and location. These data provide valuable information regarding the occurrence and epidemiology of B. bovis and B. bigemina infections in Syrian cattle, which can be employed in developing rational strategies for disease control and management.     

Serological survey of Babesia bovis and Babesia bigemina in cattle in South Africa

A total of 719 serum samples collected from clinically healthy cattle from eight provinces located in different districts of South Africa were examined by the indirect enzyme-linked immunosorbent assay (ELISA) and the standard indirect fluorescent antibody test (IFAT) to determine the serological prevalence of Babesia bovis and Babesia bigemina. The results showed that 35.3% and 39.7% of cattle were positive for B. bovis and 30% and 36.5% were positive for B. bigemina antibodies on ELISA and IFAT, respectively. Mixed infections were detected in 18.2% and 26.3% of the samples using ELISA and IFAT, respectively. Consequently, the ELISAs with recombinant B. bovis spherical body protein-4 (BbSBP-4) and B. bigemina C-terminal rhoptry-associated protein-1 (BbigRAP-1/CT) were proven to be highly reliable in the serological diagnoses of bovine babesiosis in South African cattle, as evidenced by the significant concordance rates when the results were compared to those of IFAT. Moreover, the serological prevalence was significantly different among the tested provinces, in which the ranges exhibited between 15% and 73% for B. bovis infection and between 13% and 54% for B. bigemina infection. High sero-positive rates were present in Mpumalanga and KwaZulu-Natal provinces, while the lowest rate was in the North West province. Our data provide important information regarding the current seroprevalence of bovine babesiosis in South Africa, which might be beneficial in developing rational strategies for disease control and management.     

Antibodies to mycobacteria in cattle not infected with Mycobacterium bovis

An indirect anti-IgG enzyme-linked immunosorbent assay (ELISA) using a whole cell sonicate of Mycobacterium bovis as the coating antigen, was used to detect anti-mycobacterial antibodies in cattle not infected with M. bovis. False positive M. bovis ELISA scores were produced in 6 cattle experimentally inoculated with Mycobacterium avium-intracellulare-scrofulaceum (MAIS) serovars 2, 8, 9, 14 and 18 and Mycobacterium flavescens, respectively. False positive ELISA results were also found in 39.5% of cattle from which other mycobacteria were cultured and in 56.4% of necropsied cattle with other pathological conditions. No M. bovis was cultured from these animals. Other groups of animals, with no pathological conditions, which had been tuberculin-tested negative, tuberculin-tested positive and never tuberculin tested showed positive ELISA results in 15.4%, 73.6% and 42.4% of the respective groups. The variation of these non-specific responses in uninfected cattle highlights the need for careful selection of negative controls in evaluating ELISAs for the diagnosis of bovine tuberculosis.     

Population structure of Mycobacterium bovis isolates from cattle in Mexico

The molecular fingerprints of 878 isolates of Mycobacterium bovis collected from cattle between 2009 and 2010 in different regions of Mexico were used in this study. One hundred and ninety-four spoligotypes were observed in total with a high degree of heterogeneity. Sixty-four percent of the isolates grouped into just nine spoligotypes, and 27% fell into only two spoligotypes: SB0673 and SB0669; 149 were orphan spoligotypes. The two predominant spoligotypes were found in almost all states in Mexico, especially in central Mexico, where there is a high concentration of dairy cattle; however, some spoligotypes were closely associated with restricted geographical areas. The hypothetical evolutionary relationship among spoligotypes was estimated using the spoligoforest program in the spolTools webpage. Four trees with connected components and nine unconnected nodes were found. The biggest tree had SB0140 strain as a root, suggesting this as the oldest strain in the tree. However, the relationship of this spoligotype with SB0673 and SB0669 was weak. The discriminatory power of spoligotyping for this M. bovis sample of isolates was 0.94, and the recent transmission index (RTI) 0.83, suggesting a high rate of recent transmission of some strains of M. bovis in the population. This parameter indicates that new measures are required to stop the dissemination of tuberculosis in cattle.     

Lesion development and immunohistochemical changes in granulomas from cattle experimentally infected with Mycobacterium bovis

Mycobacterium bovis, the causative agent of bovine tuberculosis, persists within granulomas. Formation of granulomas involves a complex array of immune activation and cellular migration. To examine temporal changes in granuloma development, we inoculated 32 cattle with M. bovis of deer origin. Tissues from 4 calves each were examined at 15, 28, 42, 60, 90, 180, 270, and 370 days after inoculation. Granulomas in the medial retropharyngeal lymph node were staged (I-IV) on the basis of cellular composition and the presence or absence of necrosis and peripheral fibrosis. Immunohistochemistry for inducible nitric oxide synthase (iNOS), CD68, CD4, CD8, and gamma/delta T cells was performed. Fifteen days after inoculation only stage I granulomas were seen, while between 28 and 60 days, there was a steady progression through granuloma stages such that by day 60, granulomas of all 4 stages were seen. Acid-fast bacilli were present in moderate-to-large numbers in stage I granulomas 15-60 days after inoculation. Stage IV granulomas contained large numbers of acid-fast bacteria. Abundant iNOS immunoreactivity was associated with granulomas from day 15 through day 60 but was minimal from day 90 to the termination of the experiment. The relative number of CD4+ and CD68+ cells remained constant throughout the study. In contrast, at time points >60 days, numbers of CD8+ and gamma/delta T cells diminished. Tuberculous granulomas are dynamic lesions that follow an orderly progression through disease stages. Diminished expression of iNOS and reduced numbers of CD8+ and gamma/delta T cells late in the progression of tuberculous granulomas may represent a failure of the host response to control infection.     

IL-4 and IL-10 inhibition of IFN-gamma- and TNF-alpha-dependent nitric oxide production from bovine mononuclear phagocytes exposed to Babesia bovis merozoites.

The requirement for IFN-gamma and/or TNF-alpha as co-stimulants with Babesia bovis merozoites for nitric oxide (NO) production was examined, as well as the regulatory role of IL-4 and IL-10. Purified B. bovis merozoites did not induce the production of NO in undifferentiated monocytes without addition of exogenous IFN-gamma and TNF-alpha unless the monocytes taken ex vivo were producing TNF-alpha endogenously. Under the latter condition, the NO production resulting from merozoite stimulation remained IFN-gamma-dependent. There was no evidence for endogenous synthesis of TNF-alpha in monocyte-derived macrophages (MDM), and merozoites alone were incapable of inducing TNF-alpha mRNA in MDM. However, while merozoites plus IFN-gamma induced TNF-alpha mRNA expression in MDM, NO was not produced. Both IL-4 and IL-10 inhibited expression of iNOS and production of NO in merozoite-stimulated monocytes.     

Development of a whole blood lymphocyte stimulation assay for detecting hypersensitivity to PPD in cattle experimentally infected with Mycobacterium bovis

A whole blood lymphocyte stimulation assay utilizing the uptake of tritiated thymidine was developed for the detection of Mycobacterium bovis sensitivity in cattle. Results on eight M. bovis infected animals (six to ten weeks after infection) and eight control animals show that satisfactory lymphocyte stimulation can be obtained using heparinized whole blood diluted 10-fold in tissue culture medium and cultured with purified protein derivative (PPD) for three to seven days. Infected animals exhibited significantly greater stimulation when cultured with PPD than did control animals.     

Prevalence and molecular detection of Anaplasma marginale, Babesia bovis and Babesia bigemina in cattle from Puntarenas Province, Costa Rica

A study was conducted in 2008 to determine the prevalence of Anaplasma and Babesia infections in cattle in the Puntarenas Province of Costa Rica. Blood samples were taken from a total of 449 cattle during the month of March at 30 farms in the region of Espiritu Santu, Costa Rica. Commercially available enzyme-linked immunosorbent assays (ELISA) were used to determine presence of antibodies to Babesia bigemina and Anaplasma marginale, and real-time PCR was used to determine the presence of DNA from the disease-causing organisms. The ELISA results indicated that 87.5% of the cattle sampled were positive for antibodies to A. marginale, while 59.1% were positive for antibodies to B. bigemina. The real-time PCR results showed that 235 cattle were carrying A. marginale DNA (56.9%), 6 with B. bigemina DNA (1.34%), and 2 with B. bovis DNA (0.45%).     

Molecular genotyping and epidemiology of Mycobacterium bovis strains obtained from cattle in Iran

Restriction fragment length polymorphism (RFLP) genotyping was employed to analyze the population genetics of Mycobacterium bovis in Iran. One hundred and twenty-three isolates collected from slaughtered tuberculosis-suspect cattle and one clinically asymptomatic buffalo were subjected to RFLP analysis with probes of the polymorphic GC-rich sequence (PGRS) and the direct repeat sequence (DR) using DNA digested with PvuII and AluI. All these methods detected a large homogeneous population in which only a few isolates had variant genotypes. Only AluI-based RFLPs of both the PGRS and DR sequences were able to clearly differentiate between BCG and field strains of M. bovis. As in previous reports, these findings seem to reflect a recent dispersal of one or a few strains in Iran following the substantial expansion of Holstein-Friesian cattle over the last few decades.     

Vaccination with Mycobacterium bovis BCG Strains Danish and Pasteur in White‐tailed Deer (Odocoileus virginianus) Experimentally Challenged with Mycobacterium bovis

Wildlife reservoirs of Mycobacterium bovis represent serious obstacles to the eradication of tuberculosis in domestic livestock and the cause for many faltering bovine tuberculosis eradication programmes. One approach in dealing with wildlife reservoirs of disease is to interrupt inter‐species and intraspecies transmission through vaccination of deer or cattle. To evaluate the efficacy of BCG vaccination in white‐tailed deer, 35 deer were assigned to one of three groups; one s.c. dose of 10⁷ CFU of M. bovis BCG Pasteur (n = 12); 1 s.c. dose of 10⁷ CFU of M. bovis BCG Danish (n = 11); or unvaccinated deer (n = 12). After vaccination, deer were inoculated intratonsilarly with virulent M. bovis. Lesion severity scores of the medial retropharyngeal lymph node, as well as all lymph nodes combined, were reduced in vaccinated deer compared to unvaccinated deer. BCG Danish vaccinated deer had no late stage granulomas characterized by coalescent caseonecrotic granulomas containing numerous acid‐fast bacilli compared to BCG Pasteur vaccinated or unvaccinated deer where such lesions were present. Both BCG strains were isolated as late as 250 days after vaccination from deer that were vaccinated but not challenged. In white‐tailed deer, BCG provides protection against challenge with virulent M. bovis. Issues related to vaccine persistence, safety and shedding remain to be further investigated.     

Mycobacterium bovis AN5 antigens vary in their ability to induce nitric oxide production in blood monocytes of experimentally infected cattle

Reactive nitrogen intermediates (RNI) are the principal effector molecules of activated monocyte/macrophage populations, responsible for killing and inhibiting the growth of virulent mycobacteria. In vitro nitrite production by blood monocytes of cattle inoculated with live Mycobacterium bovis AN5 was assessed from 0 day through 45 weeks post inoculation (PI). High in vitro nitrite production was observed at the 8th and 12th weeks PI in sensitized cattle but reactivity had fallen by the 20th week PI. To assess the in vitro nitrite producing ability of monocytes induced by individual polypeptides within culture filtrate antigens (CFA) of M. bovis AN5, cellular blotting was performed using peripheral blood mononuclear cells (PBMC) at the 12th week PI. It was observed that polypeptides of MW 70, 65, 60, 25, 24 and 22 kDa of CFA induced high nitrite production by blood monocytes while many polypeptides had little or no effect.     

Isolation of Mycobacterium bovis from the respiratory tracts of skin test-negative cattle.

Mycobacterium bovis was isolated from the respiratory tracts of three cattle which registered negative to tuberculin testing; no tuberculous lesions were found and the culture of lymph nodes and other tissues proved negative. One animal was from a group of five calves which had been inoculated intranasally with M bovis, and the organism was recovered once only from nasal mucus sampled 100 days after inoculation. The second animal had had contact with experimentally infected cattle which were excreting M bovis and the third was from a commercial farm. The results of ELISAS for antimycobacterial antibodies and interferon-gamma, and of lymphocyte transformation assays are presented. The animals' immune responses provided evidence that each of them had been challenged.     

Pulmonary lesions and Mycobacterium bovis excretion from the respiratory tract of tuberculin reacting cattle

Although it is generally recognised that tuberculous lesions are present in lymph nodes associated with the respiratory tract in approximately 90 per cent of reactors with confirmed infection, lung lesions are found in only 1 to 2 per cent of such cases during abattoir examination. When lung lesions are not detected, it has been claimed that such cattle are non-excretors and thus unimportant in the epidemiology of the disease. In this study the lungs of 55 reactor cattle were sliced into sections approximately 0.5 cm thick. Tuberculous lesions were evident in over 70 per cent of lungs from reactors with concurrent lesions in lymph nodes of the respiratory system. Further, M bovis was isolated from single samples of nasal and, or, tracheal mucus taken at slaughter in 19 per cent of confirmed cases. Several of these reactors had a clear tuberculin test less than six months previously indicating recent infection. This study confirms the continued importance of the infected bovine in the epidemiology and current eradication of bovine tuberculosis. It is suggested that all tuberculous cattle with lesions in respiratory lymph nodes, rather than being regarded as non-excretors, should be considered as possible excretors and thus important sources of infection for other cattle both within and between herds.     

In vivo and in vitro responses of cats sensitized with viable Mycobacterium bovis (BCG).

Cats were injected subcutaneously with viable Mycobacterium bovis (BCG), and immune responses were evaluated at various times after injection. The BCG injection produced fever, leukocytosis, neutrophilia, and lymphadenopathy of regional lymph nodes. Intradermal tuberculin injection produced responses consistent with delayed type hypersensitivity reaction in the treated cats at postinoculation day 21. Skin responses to tuberculin were not significant at postinoculation day 49. The cellular infiltrate at the tuberculin injection site at 48 hours after injection was an admixture of polymorphonuclear cells and mononuclear cells. The BCG produces strong intradermal skin responses in the cat, but the response was not long-lived as in cattle and guinea pigs. The BCG injection did not produce significant changes in the absolute total lymphocyte and absolute T-lymphocyte numbers in peripheral blood. The percentage of T-lymphocytes was significantly higher in the BCG-treated group. Differences were not observed in lymphocyte blastogenesis with tuberculin and non-specific mitogens between BCG-treated and control cats.     

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.