Genotypic diversity in field isolates of Babesia bovis from cattle with babesiosis after vaccination

Objective To determine whether particular genotypes of Babesia bovis were common to field isolates obtained from cattle properties in Queensland where the B bovis vaccine had apparently failed.
Design A comparative study of polymerase chain reaction genotypes in different populations of B bovis .
Procedure Two polymerase chain reaction assays were applied to analyse DNA extracts of B bovis vaccine (K, T and Dixie strains) and 27 field isolates from 24 properties where disease outbreaks had occurred despite the use of the vaccine. To evaluate the stability of the genotypes identified, 11 of the field isolates were inoculated into experimental cattle that had either been previously vaccinated with T strain or not vaccinated.
Results No particular genotype of B bovis was responsible for the problems observed in previously vaccinated herds. None of the isolates had genotypes identical to the vaccine strains used. No geographic trends among the genotypes were observed. Isolates that originated from the same property also had different genotypes. Blood passage of the 11 field isolates in either previously vaccinated or nonvaccinated cattle did not alter the original genotype.
Conclusion No particular genotypes identified by the Bv80 and BvVA1 polymerase chain reaction assays could be associated with vaccine failures.     

Between-herd prevalence of Mycoplasma bovis in bulk milk in Flanders, Belgium

Mycoplasma bovis (M. bovis) is a highly infectious pathogen of cattle causing pneumonia, polyarthritis, otitis, and less frequently, subcutaneous abscesses, abortions and meningitis. Ineffective drugs treatments, culling of infected cows and loss of milk production can lead to significant economic loss on dairy farms. The early detection of cows excreting M. bovis bacteria to prevent mastitis outbreaks is warranted. Reports suggest that the risk of M. bovis mastitis is higher in larger dairy herds. The objective of this study is to estimate the herd-level prevalence of M. bovis in Flanders, Belgium by culturing bulk tank milk samples taken from dairy farms. Three bulk tank milk samples per dairy herd were taken over four weeks, with collection intervals of two weeks. Culturing was done after pre-incubation using modified Hayflicks media to increase the chances of recovery of bacteria. For the identification of M. bovis, tDNA intergenic spacer PCR was used. In three herds (1.5%) of the 200 herds sampled, M. bovis was isolated from one of the three consecutive bulk tank milk samples. We conclude that in Flanders in 2009 at least 1.5% of the dairy herds had one or more cows excreting M. bovis in the milk. The frequent monitoring of bulk tank milk to detect the presence of M. bovis, especially in expanding herds on farms that often purchase replacement animals, should be encouraged in order to detect the presence of M. bovis and to monitor the success of control procedures following an outbreak of mycoplasmal mastitis in the herd.     

Increased prevalence of Mycoplasma bovis in the Netherlands.

The epidemiology, therapy, and prevention of M. bovis infections are briefly reviewed. In a survey begun in 1982, M. bovis was found frequently in the respiratory tract [corrected] of veal calves and beef cattle with respiratory problems. In replacement calves infected with respiratory disease in dairy herds, however, the organism has only been detected since 1986. Respiratory tract specimens collected from calves with respiratory disease were submitted for examination for M. bovis from 1986 to 1991 and originated from 83 herds. Mycoplasma bovis was detected in specimens from 59 of the herds, 20% of which were dairy herds and 80% fattening herds. Arthritis caused by M. bovis was observed in 12 herds until July 1991. Since 1976 when the first mastitis outbreak caused by M. bovis was diagnosed, M. bovis has caused 14 more outbreaks. The number of diseased cattle varied from 1 tot 16 per farm, and clinical signs of mastitis varied from mild to severe. In all instances the infection has been eradicated from the herds. Because M. bovis can cause great losses in intensively reared cattle herds, it is advisable to separate purchased veal calves and beef cattle from dairy cattle to prevent further spread of M. bovis.     

Risk factors for the between-herd spread of Mycobacterium bovis in Canadian cattle and cervids between 1985 and 1994.

Microorganisms of the genus Mycobacterium cause tuberculosis in many animal species including humans. Generally, Mycobacterium bovis (M. bovis) infects cattle and cervids, but it has the potential to infect virtually all species of mammals. This study examined and analysed the data from the nine outbreaks of tuberculosis in Canadian cattle and cervids from 1985 to 1994. For the purposes of this study, a positive herd was one with at least one culture-positive animal. A reactor herd had at least one animal which was positive or suspicious on a mid-cervical, comparative cervical, or gross or histopathologic test for tuberculosis. Herd classification was either reactor/positive or negative. Data for the study were collected from the outbreak records in the Regional or District offices of Agriculture and Agri-Food Canada. Logistic regression was used to study spread of tuberculosis between herds. Two risk factors were identified: increasing herd size; and, the reason why a herd was investigated as part of the outbreak. This latter factor was interpreted as a surrogate measure for the nature of contact between the study herd and other potentially infected herds in the outbreak. Increasing herd size was associated with an increased risk of being positive for tuberculosis with herds of 16-35, 36-80, and >80 animals having odds ratios of 2.9, 5.8, and 9.3, respectively, when compared to a herd size of <16 animals (p < 0.001). When compared to perimeter testing (i.e. testing herds within a specified radius of an infected herd), all other reasons for investigation had higher odds ratios (p < 0.001). These odds ratios were 57.8 for traceout herds (i.e. herds which had purchased animal(s) from a reactor/positive herd), 31.8 for herds with pasture or fence-line contact with a reactor/positive herd, and 14.9 for traceback herds (i.e. herds which had been a source of animals for reactor/positive herd(s)).     

Spatial relationship between Mycobacterium bovis strains in cattle and badgers in four areas in Ireland

We investigated whether strains (restriction fragment length polymorphism, RFLP-types) of Mycobacterium bovis isolated from badgers and from cattle clustered among and within four areas in Ireland. The spatial scan test and nearest-neighbor analysis were used as the spatial cluster-detection techniques. In addition, for each of the major strains, associations between the distance to badger setts and the "centroid" of the cattle farm were assessed in a logistic model. Overall, between September 1997 and May 2000, 316 and 287 M. bovis samples, from badgers and cattle, respectively, were strain-typed. The distribution of strains in badgers, and separately in cattle, differed among areas. Within each of the four large areas, badgers and cattle tended to have similar strains; this is consistent with the sharing of M. bovis strains within an area. In more detailed within-area analyses, some spatial clusters of M. bovis strains were detected, separately, in both cattle and badgers. Almost half of the infected badger setts with a specific strain were located outside of the "detected" clusters. There was no association between the number of infected badgers with a specific M. bovis strain within 2 or 5 km distances to cattle herds, and the risk of the same strain in cattle. We speculate about the dynamic nature of badger movements, as an explanation for the absence of more clusters of most of the strains of M. bovis isolated from badgers, and its impact on trying to study transmission of M. bovis between cattle and badger.     

The seroprevalence of Mycoplasma bovis in lactating cows in Switzerland, particularly in the republic and canton of Jura]

Sporadic cases of infection with Mycoplasma bovis have been observed in Switzerland since 1983. However, five severe outbreaks of endemic mastitis in a geographically distinct, small region (Canton du Jura) during the period 1995 to 1997 prompted the present investigation on the seroprevalence of infection with M. bovis among milking cows in Switzerland. A commercially prepared indirect enzyme immunoassay was used. Among a stratified random sample of 118 herds of milking cows in Switzerland, at least one positive animal was detected in 56 (47%) of the herds, whereas 6.1% of the 1816 individual animals tested positive. An epidemiological study was performed in the Canton du Jura region among 51 herds in order to assess the importance of management factors in the spread of M. bovis infection. The herd-level prevalence was 78%, and the seroprevalence at the level of the 1354 individual animals tested was 13.4%. A multivariate analysis of possible risk factors showed purchase of animals to be the only variable significantly associated with serological status of the herd with an "odds ratio" of 10.8.     

Epidemiological study of risk factors for Mycoplasma bovis infections in fattening calves

To establish the role of Mycoplasma bovis as an agent of respiratory disease in fattening calves, an epidemiologic study was undertaken. A recently validated commercially available ELISA was used to diagnose M. bovis infection by seroconversion in paired sera obtained for each animal at entry in the fattening herd and at follow-up seven weeks later. Management data as well as relevant clinical and epidemiological variables were prospectively recorded. The overall seroconversion rate observed among the 415 calves in 23 fattening herds on 13 farms was 54.7%. Significant risk factors for seroconversion were the mixing of fattening herds of different age groups (risk ratio RR 1.70, 95% confidence interval (CI) 1.48 to 1.96), and the presence of at least one seropositive animal in the fattening herd (RR: 2.02; CI: 1.69 to 2.40). The proportion of clinical episodes of respiratory disease attributable to M. bovis infection was 50.3%. The average weight gain during the observation period was reduced by 7.6% in seroconverting calves and these animals had about 2 times more antibiotics prescribed by a veterinarian than calves remaining negative for M. bovis throughout follow-up (RR 1.83). Maternal antibodies against M. bovis were detected in 39% of newborn calves born from seronegative cows and had a half-life of 20 days, potentially limiting the usefulness of vaccines against M. bovis in this age group.     

A field study of Mycoplasma bovis infection in cattle

After an outbreak of mastitis in cattle caused by Mycoplasma bovis a study was made in 5 herds with recent cases (principal herds) and in 4 control herds. In the principal herds, M. bovis was isolated from milk samples, nasal swabs, and from one vaginal swab. M. bovis was also isolated from nasal swabs of calves in 2 of the 4 control herds, whereas all milk samples and vaginal swabs from the control herds were negative. Evaluation of serum antibody titres to M. bovis among non-mastitic animals of 3 principal herds and 1 control herd showed no difference in distribution of the titre values, which generally were low. However, cows excreting M. bovis in the milk had high antibody titres. The way of introduction to the herds and the spread of the infection within the herds could not be established by the study, which was supplemented by a DNA restriction fragment analysis of a number of M. bovis isolates.     

Immunogenicity of Moraxella bovis hemolysin

Anti-Moraxella bovis hemolysin activity was observed in 35 cattle exposed to field infections of infectious bovine keratoconjunctivitis (IBK). All cattle in infected herds seroconverted with respect to antihemolysin whether or not clinical IBK was noted. Cattle previously exposed to IBK possessed higher antihemolysin titers than did younger, nonexposed cattle. Antihemolysin activity was noted in bovine sera up to 7 years after exposure to IBK. Sera from experimentally infected calves were found to possess antihemolytic activity against all 33 strains of M bovis tested. Antihemolytic activity could be demonstrated in random-bred mice inoculated with whole doxycycline-treated M bovis, frozen or lyophilized whole M bovis, and membrane fractions treated with sodium lauryl sarcosinate, Triton X 100, and Triton X 100 + EDTA, but not with formalin-treated whole M bovis.     

Characterisation and quantitation of pilus antigens of Moraxella bovis by ELISA

Hyperimmune serums raised in rabbits to purified pili from 9 Australian and 2 American strains of Moraxella bovis from infectious bovine keratoconjunctivitis (IBK) affected herds were used to study the degree of binding between combinations of antigen and antiserum in a conventional enzyme linked immunosorbent assay (ELISA). With the aid of appropriate absorption tests major antigenic differences among pili were found permitting 6 distinct serogroups to be recognised. Further, production of specific antiserums to representative strains of each serogroup in goats facilitated the development of a double antibody sandwich ELISA which could be used to quantitate pilus expression of a given strain of M. bovis, or to differentiate pilus serogroups of 22 strains of M. bovis obtained from a total of 12 Australian herds. Most isolates were found to belong to serogroups designated IV and V. One strain from the United States of America showed total homology with Australian serogroup IV while the other showed some cross-reactivity with serogroups V and VI.     

Bovine cysticercosis in Denmark. A study of possible causes of infection in farms with heavily infected animals

Epidemiological studies were made on 14 farms from which, during a 2 year period, 38 cattle had been condemned at slaughter, due to massive infections with Cysticercus bovis. By on-site investigations and interviews, attempts were made to identify the transmission routes of Taenia saginata eggs from human faeces into the environment and further on to cattle. The most frequent sources of infection were found to be sludge from septic tanks illegally applied on pasture or crops, in some cases after having been mixed with animal slurry. Animals in permanently housed herds were infected through the fodder or by contamination of the indoor environment by such slurry containing Taenia eggs. Other herds were infected by grazing pastures in close proximity to municipal sewage treatment plants. In contrast to earlier Danish observations, application on farmland of sewage sludge from municipal treatment plants was not involved in any of the reported outbreaks. This apparent change coincides with the implementation of more restrictive legislation for the agricultural use of sewage sludge in Denmark.     

Pathologic findings and association of Mycobacterium bovis infection with the bovine NRAMP1 gene in cattle from herds with naturally occurring tuberculosis

OBJECTIVE: To determine necropsy and Mycobacterium bovis culture results in cattle from herds with tuberculosis, the role of the bovine NRAMP1 gene in resistance and susceptibility to infection with M bovis, and the association between magnitude of the tuberculous lesions and various types of M bovis isolates. ANIMALS: 61 cattle from herds with tuberculosis in Texas and Mexico. PROCEDURE: 61 cattle were evaluated by necropsy; 59 had positive and 2 had negative caudal fold tuberculin intradermal test (CFT) results. Thirty-three cattle with positive CFT results were genotyped to evaluate polymorphism of the 3' untranslated region of the bovine NRAMP1 gene, using single-stranded conformational analysis, 9 were resistant to M bovis with no tuberculous lesions and negative M bovis culture results, and 24 were susceptible with tuberculous lesions and positive M bovis culture results. Isolates of M bovis were analyzed by restriction fragment length polymorphism (RFLP) on the basis of IS6110 sequences and direct-repeat fingerprinting patterns. RESULTS: 21 (35.6%; 21/59) cattle with positive CFT results had tuberculous lesions or positive culture results; in addition, 1 of 2 cattle with negative CFT results had tuberculous lesions and positive culture results. Tuberculous lesions were most common in the thorax (35/63; 55.5%) and lymphoid tissues of the head (10/63; 15.9%). Tuberculous lesions varied from 1 to 11/animal; 8 of 21 (38.1%) had solitary lesions. Associations were not found between resistance or susceptibility to infection with M bovis and polymorphism in the NRAMP1 gene or between the magnitude of the lesions and various RFLP types of M bovis isolates. CONCLUSIONS AND CLINICAL RELEVANCE: The NRAMP1 gene does not determine resistance and susceptibility to infection with M bovis in cattle.     

The epizootiology of tuberculosis of cattle in the Federal Republic of Germany

Mycobacterial strains from different outbreaks of tuberculosis of cattle in Germany from 1996 to 2001 were differentiated by two molecular biological methods (Spoligotyping, RFLP IS6110). The causative agent was in one case Mycobacterium (M.) africanum, in 10 cases M. bovis and in 17 cases M. bovis ssp. caprae, respectively. The results of the molecular biological methods are discussed from the perspective of epizootiology and the particular importance of infections by M. bovis ssp. caprae emphasized. Direct contact of the animals, purchase from infected stocks, infected zoo animals and wildlife, as well as livestock handlers are discussed as possible sources of infection.     

Serologic cross-reactivity of Australian Moraxella bovis to vaccinal bacterin strains as determined by competitive ELISA

OBJECTIVE: To conduct a serologic survey and define pili antigenic variability via the serologic cross-reactivity of Moraxella bovis isolates from naturally occurring infectious bovine keratoconjunctivitis (IBK) outbreaks in Australia. This project applies to the development of an M bovis pili-based vaccine targeting Australian strains originating from intensive cattle producing regions. PROCEDURE: Ocular swabs were collected from cattle affected with clinical signs of IBK from 25 veterinary practices. Standard criteria were used to identify 70 M bovis. Pure, piliated isolates were evaluated with a modified competitive enzyme-linked immunosorbent assay (ELISA) for cell-bound M bovis pili to determine their serologic cross-reactivity with pili of vaccinal bacterin strains EPP63, FLA64, and SAH 38. RESULTS: Sixty-four percent (45/70) of M bovis isolates demonstrated homologous pili antigens to a vaccinal strain. M bovis isolates homologous to one of the three vaccinal strains were obtained in 77% (34/44) of IBK outbreaks sampled. No IBK outbreak had isolates homologous to more than one vaccinal strain; however, 29% (10/34) of outbreaks with a cross-reacting strain had non-cross-reacting strains as well. CONCLUSION: The similar prevalence of pilus antigen homology to strain FLA64 was observed with isolates derived from NSW, Tasmania, and Victoria, compared with results of prior smaller serologic studies, suggests that the common pilus antigens in M bovis within Australia have been relatively stable over the last 20 years. The prevalence of a limited number of pilus antigens in M bovis suggest that the application of a vaccine containing the bacterial strains EPP63, FLA64, and SAH38 may provide a useful management tool for reducing production losses associated with IBK in Australia.     

Studies of bovine Mycoplasma mastitis. 4. Serological classification of Mycoplasma strains isolated from 3 stocks

Thirty-two isolates from altered milk samples taken from cows with mastitis of two herds were serologically tested by the WHT against rabbit hyperimmune sera of 16 reference and type strains. The strains tested were associated and grouped in the following way: 16 M. bovis, nine A. laidlawii, and seven A. axanthum. The classification of on M. bovis strain was confirmed by SHT. Five isolates from another stock, with biochemical properties related to the family of mycoplasmataceae, were not serologically identified.     

The emergence of Mycobacterium paratuberculosis in farmed deer in New Zealand - a review of 619 cases

AIM: To review cases in which Mycobacterium paratuberculosis was identified in farmed deer in New Zealand. METHODS: Case histories were reviewed where M. paratuberculosis was identified in deer by either culture or a polymerase chain reaction (PCR) test using primers from IS900. RESULTS: Between 1986 and 2000, M. paratuberculosis was identified by bacterial culture and/or PCR in 619 farmed deer from 299 herds, representing approximately 6% of deer herds in New Zealand. Over 85% of cases were identified during the last 6 years. In 60% of the infected herds, only one infected animal was identified. The maximum number of cases identified in a single deer herd was 47, and these were identified over a period of 8 years. Only 36 (5.8%) cases came from clinically affected animals identified on farms by veterinarians. The majority (89.7%) of the 619 cases were identified from lesions in mesenteric lymph nodes, including the ileocaecal lymph nodes, identified at meat inspection as being macroscopically either typical or equivocal of bovine tuberculosis (M. bovis). While the overwhelming majority of lesions were identified in mesenteric lymph nodes, M. paratuberculosis was also identified in 27 lesions in lymph nodes of the head, especially the retropharyngeal lymph node. CONCLUSIONS: The figures presented underestimate the true prevalence of infection with M. paratuberculosis, especially since not all suspect cases were submitted for culture or PCR. However, they do show that M. paratuberculosis appears to be spreading in farmed deer in New Zealand and highlight the possibility that Johne's disease is emerging as a potential major problem affecting this species. Identification of the organism by bacterial culture or PCR is required in many cases to distinguish lesions in mesenteric lymph nodes and lymph nodes of the head caused by M. paratuberculosis from those caused by M. bovis and M. avium.     

Failure to identify non-bovine reservoirs of Mycobacterium bovis in a region with a history of infected dairy-cattle herds

The State of Texas had the most (cumulative) tuberculous cattle herds of any state in the United States during the decade ending in 1997. Of the cumulative 18 infected herds in Texas, 12 herds were concentrated in El Paso County (designated the 'El Paso milkshed'). To identify whether non-bovine reservoirs were a source of Mycobacterium bovis infection of cattle in this region, an investigation was conducted on the premises of 14 dairy herds (12 tuberculous and 2 non-affected herds) between May 1995 and June 1997. None of the 670 mammalian, avian and environmental (soil, water and air) samples collected and cultured from the premises of these herds was positive for the presence of M. bovis. None of the 119 human urine samples obtained from employees of these dairies was culture positive for M. bovis. Of 124 dairy-farm workers with tuberculin skin-test results, 48 showed positive test results. There was, however, no difference in percentages of positive skin-test results between farms without, and farms having, bovine tuberculosis within the last two years or longer. The percentage of positive reactions did not increase with length of time employed at a dairy with a history of confirmed tuberculosis. These findings suggest that non-bovine reservoirs appear not to be a factor responsible for tuberculosis of cattle in the El Paso milkshed.     

Prevalence of mycoplasmas in the respiratory tracts of pneumonic calves.

The prevalence of mycoplasmas in the respiratory tracts of 148 pneumonic calves originating from 25 herds in the Netherlands is reported. Four types of culture media were used to isolate mycoplasmas: solid modified Edward medium, 2 types of Friis media, and A7B differential agar medium. Mycoplasmas were isolated both from nasal swab specimens and lung lavage fluids collected from live calves and from nasal mucosa and lung tissue specimens collected post mortem. All of the mycoplasma strains isolated could be identified as either Ureaplasma diversum (isolated from 80% of 25 herds), Mycoplasma dispar (92%), M. bovirhinis (88%), M. bovis (20%), M. bovigenitalium (4%), M. arginini (16%), or M. canis (12%). Isolation rates of M. dispar and U. diversum were considerably higher from lung lavage fluids than from nasal swab specimens. M. bovis was detected only in fattening herds and not in dairy herds. The respiratory tracts of 75% of the calves examined contained at least 2 mycoplasma species. In total, 25 different combinations of mycoplasma species were detected in specimens collected from noses and lungs. The pathogenic species U. diversum and M. dispar had not been isolated before in the Netherlands.     

The epizootiological significance of positive bacteriological findings on Mycobacterium tuberculosis and Mycobacterium bovis in humans

The relation between the active form of tuberculosis in persons working in agriculture and incidence of tuberculosis in cattle was analyzed in 1974 to 1978, i.e. in the period after the elimination of bovine tuberculosis in Czechoslovakia (in 1968). M. tuberculosis was isolated in 15 cases and M. bovis in four cases of persons employed by the farms on which the Regional Hygienic Station, Brno, was responsible for the microbiological diagnostics of tuberculosis. Direct contact with animals was demonstrated in eight patients; M. tuberculosis was isolated from seven of these patients and M. bovis from one. Seven cattle herds were exposed to spontaneous infection by M. tuberculosis and in one of them tuberculosis was not demonstrated during complex examination. In three herds the examination revealed only a sensitivity of cattle to mammalian tuberculin. In other three herds tuberculosis was detected by allergic tests, patho-anatomic examination and bacteriological examination. M. tuberculosis in cattle was detected in two herds. The occurrence of bovine tuberculosis caused by a cattle tender with a positive finding of M. bovis in sputum was demonstrated in one herd. Virulence for the tested cattle was found in one strain (isolated from a mesenterial lymph node of cattle) of the four strains of M. tuberculosis used for the experimental infection of 17 animals. On the other hand, in three strains of M. tuberculosis, trials with experimental infection demonstrated only allergy to mammalian tuberculin and changes at the sites of subcutaneous inoculation of mycobacteria of regressive nature; these mostly disappeared within 90 days from infection.     

The use of pulsed-field gel electrophoresis to investigate the epidemiology of Mycoplasma bovis in French calf feedlots

Mycoplasma bovis is a major cause of respiratory outbreaks in cattle feedlots. In this study pulsed-field gel electrophoresis (PFGE) was used to trace field strains and provide information on M. bovis patterns of spread in calf feedlots. The suitability of KpnI, MluI and SmaI restriction enzymes was assessed on different sets of strains. The discriminative power of the first two enzymes was first assessed using 28 epidemiologically unrelated strains; stability was 100% on multiple isolates from in vivo experimental infection. Thirty-nine field isolates from six feedlots were then evaluated. In contrast to the unique fingerprints displayed by the unrelated strains, the isolates from the feedlots showed identical patterns at the time of the outbreak of respiratory disease and 4 weeks later. The PFGE typing results suggest that M. bovis strains follow a clonal epidemic spread pattern at the herd level and that the same strain persists in calves of the herd after the clinical signs have disappeared.