Field evaluation of tracer sheep for the detection of early natural infection with Mycobacterium avium subsp paratuberculosis

OBJECTIVE: To determine whether tracer sheep could be used to detect S strain Mycobacterium avium subsp paratuberculosis on pasture, and to provide further insight into the early stages of infection. DESIGN: A field study on two farms in an endemic area for ovine Johne's disease in New South Wales. Procedure Lambs, weaners and adult ewes were introduced to pasture with varying amounts of M. a. paratuberculosis contamination and monitored using skin tests, gamma interferon assay, faecal culture and serial necropsy of small groups for up to 15 months after first exposure. RESULTS: Culture from tissues was the most sensitive method for detecting early infection in sheep after natural exposure to S strain M. a. paratuberculosis. The organism was detected in at least one introduced sheep from every exposed group, 6 to 12 months after first exposure. Histopathological lesions were detected in only 17% of culture-positive sheep, and only after at least 8 months of exposure. Similarly, antemortem diagnostic tests had low sensitivity during the early stages of naturally acquired infection. There was no evidence of any differences in infection rate between sheep first exposed as neonates, as weaners or as adults. A higher proportion of lambs born to ewes from an infected flock were infected than lambs suckling uninfected ewes introduced to the same infected environment, and infection was detected earlier in these 'resident' lambs. CONCLUSION: These findings indicate that groups of unexposed 'tracer' sheep, tested by culture of tissues at slaughter 6 to 12 months after first exposure, might be a useful way to assess pasture infectivity in control programs for ovine Johne's disease.     

Immunohistochemical expression of interferon-γ in different types of granulomatous lesions associated with bovine paratuberculosis

Animals infected with Mycobacterium avium subsp. paratuberculosis (Map) show a variety of lesions, from focal forms, seen in subclinical stages to diffuse lesions in clinical cases. The purpose of this study was to evaluate the local expression of IFN-γ by immunohistochemistry in relation with the type of lesion in naturally Map-infected cows. The number of immunolabelled cells, −the majority morphologically consistent with lymphocytes-, was higher in focal and diffuse paucibacillary forms than in diffuse multibacillary lesions, where they appeared closely related to epithelioid cells. Diffuse multibacillary lesions had the lowest numbers, but higher than controls, and positive cells were intermingled among the macrophages. The peripheral IFN-γ production was higher in all Map infected cows and a positive correlation was found with the number of immunolabelled cells in the intestine. The findings of this study show that IFN-γ would play a role in the development of the different types of lesions in paratuberculosis, and also points out the importance of adequate sampling of lymphoid tissue containing samples when studying the local immune response in which IFN-γ expression may be involved, especially in cases where focal lesions are present.     

Enhancement of the interferon gamma assay to detect paratuberculosis using interleukin-7 and interleukin-12 potentiation

A limitation to the widespread application of interferon gamma (IFN-γ) response assays has been logistical difficulties, as they must be performed within hours of blood collection. Detection of specific IFN-γ responses to Mycobacterium avium subspecies paratuberculosis (MAP) as part of the cell-mediated immune response of ruminants with Johne's disease could aid in diagnosis and control programs. In this study, a modified protocol was developed in which cultures were supplemented with interleukin (IL)-7, a survival factor required to maintain resting T cells, alone or in combination with IL-12 to potentiate IFN-γ responses and extend blood storage time. The combination of IL-7 and IL-12 was synergistic, giving IFN-γ responses greater than with IL-12 alone, for sheep blood stored up to 2 days. In a cohort of naturally infected sheep, the same number of animals was identified as test positive using the modified assay (performed after 2 days with IL-7/IL-12 supplementation) as the standard IFN-γ assay performed on the day of blood collection. The modified assay offered greatest advantage in the detection of early stages of paratuberculosis infection, for sheep with low grade and paucibacillary lesions, and at early time points post-infection. The potentiation protocol allowed for practical shipment of blood samples from farm to laboratory, extending permissible transit times and applicability of IFN-γ testing to detect Johne's disease.     

Immuno-electromicroscopic approach for the study of neural stem cell niches

Lymphoproliferative response (LPR) was studied in 19 lambs orally infected (Group I) with Mycobacterium avium subsp. paratuberculosis (MAP) with in vitro lymphocyte stimulation test using MTT dye reduction assay. The non-specific LPR against Con A and specific LPR against sonicated antigen and johnin PPD (purified protein derivatives) were estimated on preinfection (0 day) and various days postinfection period (15 to 330 dpi) in the animals, which were classified according to histological and bacteriological evidence of paratuberculosis infection. Of the two antigens used, johnin PPD was found to be superior in terms of consistency and uniformity of response over an observation period of about a year. Significantly (P < 0.05) higher LPR were observed in the infected sheep during postinfection period, as compared with preinfection values and values from uninfected control sheep. It was evident from the present study that the LPR in histologically infected animals fluctuated during the long course of infection and had a definite relationship with the gut pathology and the mycobacterial load. The LPR were stronger but variable in sheep with grades 1, 2 and 3 lesions (paucibacillary) and increased progressively from 30 dpi onwards. The sheep with the advanced lesions (grade 4, multibacillary) showed progressive decline in LPR till 120 dpi after initial stronger response at 30 dpi. Most of the animals were detected by LPR before initiation of faecal shedding of MAP. The results suggested that repeated testing was required while screening an infected flock for detecting most of the positive animals.     

Paratuberculosis in sheep: its possible role in the epidemiology of paratuberculosis in cattle

A total of 50 sheep originating from 15 Dutch farms with a known paratuberculosis infection in their cattle herd, but with no history of paratuberculosis infection in their sheep flock, were examined for infection with Mycobacterium avium subsp. paratuberculosis (Map). The sheep had been grazing on the same pastures as the cattle or on pastures fertilised with manure from these cows. The sheep were screened for paratuberculosis by serum biochemistry, serology and intradermal skin tests. At necropsy they were examined macroscopically, microscopically and bacteriologically for paratuberculosis.From 10 sheep, originating from eight flocks, Map could be isolated from various tissues but not from the intestinal contents, after an incubation period of 2.5-4 months. Six of these culture-positive sheep had no macroscopic signs of paratuberculosis at necropsy. Seven sheep were Map culture negative but showed macroscopic and microscopic lesions consistent with a paratuberculosis infection. Results of serology and skin tests did not correlate with the results of bacteriological culture. Serum concentrations of calcium, albumin and total protein of the infected, suspected and negative sheep were not different. These results indicate that a substantial number of the sheep examined were infected with Map. Even though this bacterium was not isolated from their faeces, the possibility that these sheep could have been shedding Map with their faeces below detection level or at a later stage of the disease cannot be eliminated. Map infected sheep should, therefore, be considered as a possible factor in the epidemiology of with Map infected cattle herds in The Netherlands. At necropsy bacteriological culture of Map should be performed on a routine basis to improve the diagnosis of paratuberculosis in sheep.     

Experimental infection with Mycobacterium caprae in goats and evaluation of immunological status in tuberculosis and paratuberculosis co-infected animals

Tuberculosis in goats (caused by Mycobacterium caprae and M. bovis) has become a significant concern in recent years because of its high prevalence in certain caprine herds in Spain and other European countries, and also due to the potential transmission to other animals and human beings. In the present study, a transthoracic model of tuberculosis infection was performed on goats. Animals were selected based on the serological response used to detect paratuberculosis in goats (negative and positive results). The kinetics of the immune response was evaluated using the interferon-γ (IFN-γ) assay, skin tests and serology of paratuberculosis during nine months post-challenge. At the end of the study the animals were necropsied, tuberculosis-lesions were scored and culture (M. caprae and M. avium subsp. paratuberculosis) was performed to determine the true infection status. Animals were positive to the IFN-γ assay 15 days post-challenge and the values were fluctuating throughout the study. A varied performance of the assay was observed between tuberculosis and tuberculosis-paratuberculosis mixed infection regarding both the number of positive results and the OD values obtained after stimulation with bovine and avian PPDs. Furthermore, the single intradermal comparative cervical tuberculin test did not detect all M. caprae-infected animals. At necropsy, a positive correlation between pathology score and bovine PPD specific IFN-γ response was found.     

Effects of helminth infection on the pre-weaning production of ewes and lambs: evaluation of pre- and post-lambing drenching and provision of safe lambing pasture

The pre-weaning production of ewes and lambs in spring on pastures grazed during the previous autumn and early winter by either weaner sheep, adult sheep, or cattle was investigated together with estimating the benefits of pre- and post-lambing drenches to ewes. These treatments were compared with one in which parasitism was uncontrolled in ewes lambing on pastures grazed previously by untreated weaner sheep, and another where parasitism was suppressed by 2-weekly drenching of the weaner sheep and also of the ewes and their lambs. Prior grazing by cattle effectively eliminated infection of pastures with intestinal Trichostrongylus and Nematodirus spp, but less so for Ostertagia spp. Worthwhile reductions in contamination were also achieved by grazing by adult sheep compared with grazing by undrenched weaners. Despite differences in the parasitological status of the pastures, there were no indications that pre-weaning growth rates of lambs were affected. However, wool growth in ewes was reduced by 10 to 20% by parasite infection. Wool growth of ewes on pastures grazed by cattle during the pre-experimental period exceeded that on any other treatment, and was significantly greater than that of ewes on pastures grazed by undrenched weaners. There was no production benefit in giving a pre-lambing drench to ewes on plots contaminated by weaners, or in giving the additional post-lambing drench to ewes grazing on plots contaminated by weaners, adult sheep or cattle.     

Antigenic profiles of recombinant proteins from Mycobacterium avium subsp. paratuberculosis in sheep with Johne's disease

Methods to improve the ELISA test to detect Mycobacterium avium subsp. paratuberculosis have been explored over several years. Previously, selected recombinant proteins of M. avium subspecies paratuberculosis were found to be immunogenic in cattle with Johne's disease. In the present study, antibody responses of infected and healthy sheep were evaluated using 18 purified recombinant proteins in an ELISA-based format for the serodiagnosis of ovine paratuberculosis. These selected recombinant proteins represent heat shock proteins, hypothetical proteins and cell surface proteins of M. avium subsp. paratuberculosis. Whereas, Map0862 (a gene uniquely present in M. avium subspecies paratuberculosis) and Map3786 encoded protein solicited the strongest antibody response in infected sheep. The protein encoded by Map2116c showed the weakest antibody response among the animals tested. Although none of the recombinant proteins detected all 11 infected sheep singly, antibodies to Map0862 were detected in 9 of 11 (81%) infected sheep. Furthermore, ovine responses to these selected antigens were assessed temporally over the course of 1 year during which we found a spiking effect rather than an incremental increase of antibody reactivity. This study evaluated multiple M. avium subsp. paratuberculosis recombinant proteins in an ELISA-based format for sheep.     

Experimental infection of a bovine model with human isolates of Mycobacterium avium subsp. paratuberculosis

Mycobacterium avium subsp. paratuberculosis (Map), the etiologic agent of Johne's disease (JD) in ruminants, has been implicated in the pathogenesis of Crohn's disease (CD) in humans. We developed a bovine ileal cannulation model to facilitate comparison of the immune response to Map and the mechanisms of pathogenesis in cattle and humans. Initial studies showed a T cannula could be maintained for up to a year in calves without inducing inflammation or adversely affecting intestinal function. Map introduced through the cannula established a persistent low level of infection without inflammation. Infection elicited an immune response to Map antigens detectable by flow cytometry. Further studies now show the cannulation model can be used with cows during the later stage of infection, affording access to the target tissue at all stages of infection. The studies also revealed no difference in infectivity or immunogenicity of isolates of Map obtained from cattle or humans with CD. Comparison of the immune response to Map during the early and late stages of infection using PCR, flow cytometry and QRT-PCR, showed the immune response early in the disease process is dominated by CD4 T cells. A CD8 response is delayed but comparable at later stages of infection. Genes for pro-inflammatory cytokines IFN-γ and the recently identified genes encoding IL-17 and IL-22 are up regulated in infected animals. These findings reveal that both human and bovine isolates of Map can establish infection and induce similar immune responses in a bovine model. They also reveal the cytokine responses elicited in cattle are similar to those implicated in CD pathogenesis.     

Vaccination against paratuberculosis of lambs already infected experimentally with Mycobacterium avium subspecies paratuberculosis

OBJECTIVE: To assess the protective value of a live-attenuated vaccine in sheep already exposed to Mycobacterium avium subsp paratuberculosis and to investigate the progression of a systemic immune response in experimentally infected sheep. STUDY DESIGN: Twenty-eight lambs, aged 1 to 1.5 months, were dosed via stomach tube with approximately 4.4 x 10(8) M a paratuberculosis organisms. Two weeks later, 14 of these 28 animals received subcutaneous injections of 1 mL of a live-attenuated vaccine. Thirteen additional lambs were neither dosed nor vaccinated (negative controls). Antigen-induced production of IFN-gamma in blood, and antibody concentrations in serum were sequentially monitored in vaccinated, unvaccinated and control animals for 1 year. Each sheep was examined for infection by an IS900-based PCR test on samples of ileum and ileocaecal lymph node and histological examination at the time of necropsy. RESULTS: Seven of 14 unvaccinated and two of 14 vaccinated sheep developed clinical paratuberculosis that was later confirmed by histological examination and/or the IS900-based PCR test. The granulomatous inflammation in the jejunal and ileal mucosa was less severe in vaccinated than in unvaccinated sheep. Acid-fast organisms were detected only in the unvaccinated group. The PCR assay on ileal samples gave positive reactions in two vaccinated and eight unvaccinated sheep. Both the antibody response and IFN-gamma response were detected earlier and were more substantial in vaccinated than in unvaccinated sheep. Furthermore, in experimentally infected but unvaccinated sheep, the IFN-gamma concentrations were higher in those animals without acid-fast organisms than in those with them. CONCLUSIONS: Vaccination of lambs with live-attenuated vaccine 2 weeks after oral inoculation with M a paratuberculosis stimulated the host response against the organism and led to a reduced mycobacterial burden. The diminished IFN-gamma responses in experimentally infected sheep with acid-fast organisms suggest a positive relationship between the magnitude of the systemic cell-mediated immune response and an animal's ability to control infection.     

Demonstration of lateral transmission of scrapie between sheep kept under natural conditions using lymphoid tissue biopsy

Scrapie free adult sheep were introduced to a sheep flock specifically maintained to maximise scrapie infection. Native born sheep of the highly susceptible VRQ/VRQ genotype in this flock show highly efficient transmission, evidenced by 100% infection, with an age at death of less than 2 years. Infection in introduced sheep was identified by biopsy of tonsilar and nictitating membrane lymphoid tissue. Progeny of these sheep were monitored and clinical disease confirmed by examination of the brain using routine diagnostic methods. Na?ve sheep of New Zealand origin introduced to the flock in adulthood became infected, demonstrating that lateral transmission had occurred. Lambs born to introduced ewes became infected and died at the same age as lambs born to native ewes, consistent with lateral transmission of scrapie to lambs. Although maternal transmission cannot be totally excluded for the lambs in this study, the data are consistent with lateral transmission being the most important means of spread leading to the high incidence of scrapie observed in this flock.     

Rapid NK-cell activation in chicken after infection with infectious bronchitis virus M41

Natural killer (NK) cells are cytotoxic lymphocytes and play an important role in the early defence against viruses. In this study we focussed on NK cell and interferon (IFN) responses after infection with infectious bronchitis virus (IBV). Based on surface expression of CD107+, enhanced activation of lung NK cells was observed at 1 dpi, whereas in blood prolonged NK-cell activation was found. IFN-α and IFN-β mRNA and proteins were not rapidly induced whereas IFN-γ production in lung, measured by Elispot assay, increased over time at 2 and 4 dpi. In contrast, IFN-γ production in blood was highest at 1 dpi and decreased over time down to levels comparable to uninfected birds at 4 dpi. Collectively, infection with IBV-M41 resulted in activation of NK cells in the lung and blood and rapid production of IFN-γ and not IFN-α and IFN-β compared to uninfected birds.     

Establishment of a maedi-visna-free flock after the purchase of infected sheep

Maedi-visna (MV) infection was detected in a cohort of 68 purchased ewes, one of several groups of sheep introduced to a farm after the previous stock had been culled with suspected foot-and-mouth disease in 2001. Except for short periods totalling six to seven weeks when the sheep co-grazed with 13 ewe lambs and ram lambs, the infected cohort was kept separate from other sheep on the farm over a total of 21 months. During this period two crops of lambs were reared from the infected ewes. All the lambs were fattened and killed, and all ewes were culled after the second crop of lambs had been weaned. Subsequent serological testing of the remaining sheep on the farm confirmed the elimination of MV infection from the flock, leading to its acceptance in the Maedi Visna Accreditation Scheme of the Scottish Agricultural College's Sheep and Goat Health Schemes.     

Effect of passive transfer of maternal immune components on infection with ovine herpesvirus 2 in lambs

OBJECTIVE: To define the role of passively tranferred immunity in protection against early infection with ovine herpesvirus 2 (OvHV-2) in lambs. ANIMALS: 15 adult sheep and 34 lambs. PROCEDURES: 2 groups of animals were used, including 15 lambs born to OvHV-2-free ewes and 19 lambs born to OvHV-2-positive ewes. After nursing colostrum, all lambs and their dams were introduced into a flock positive for OvHV-2. Blood was obtained from the lambs every 2 weeks and examined by PCR assay and competitive inhibition ELISA. RESULTS: None of the animals had positive results by PCR analysis for samples obtained approximately 2 weeks after introduction into the flock. In the group of lambs from OvHV-2-infected ewes, 5 of 19 had positive results at 1 month of age and 17 of 19 by 5 months of age. In the group of offspring from OvHV-2-negative ewes, only 1 of 15 had positive results at 1 month of age, and the number reached 12 of 15 by 5 months of age. All lambs in both groups had positive results by 6 months. An active antibody response to the virus was detected in animals within 3 weeks after viral DNA became detectable in the blood. CONCLUSIONS AND CLINICAL RELEVANCE: Analysis suggests that passively transferred immunity does not play an important role in the delay of infection with OvHV-2 in lambs. Age also does not seem to influence susceptibility. The rate of infection in young lambs may simply be a reflection of the intensity of viral exposure in their environment.     

甘肃高山细毛羊枯草季放牧与暖棚舍饲饲养对比试验

枯草期选甘肃高山细毛羊成年母羊180只,后备母羊38只,羔羊40只,进行为期171 d的放牧与舍饲对比试验。结果表明:试验组(暖棚舍饲)平均每只成年母羊、后备母羊和羔羊体质量分别增加5.1、9.8和6.9 kg,增幅为:11.72%、30.81%和32.86%;对照组(放牧)每只成年母羊、后备母羊和羔羊体质量分别平均下降7.3、5.0和1.0 kg;试验末,试验组成年母羊和后备母羊体质量极显著高于对照组(P0.01),试验组羔羊体质量显著高于对照组(P0.05)。羊毛长度试验组成年母羊、后备母羊和羔羊羊毛每只分别平均增长3.9、3.8和2.7 cm,试验组成年母羊和后备母羊比对照组提高10.4%和16.0%,羔羊比对照组降低11.2%,试验组和对照组差异不显著(P0.05)。试验组各年龄段产羊毛量分别达5.24、4.56和3.79 kg,比对照组分别提高60.24%、73.38%和49.80%,差异极显著(P0.01)。试验组纯收入分别为成年母羊5.35元/只,后备母羊45.65元/只,羔羊59.57元/只,对照组由于掉膘损失严重,成年母羊和后备母羊分别损失28.32和13.05元/只,羔羊收益29.3元/只。     

Experimental infection of weaner sheep with S strain Mycobacterium avium subsp. paratuberculosis

We sought to determine whether infection of recently weaned 12-16-week-old Merino lambs with an Australian S strain M. a. paratuberculosis, at doses consistent with natural exposure, could be detected in the first few months post-inoculation. Such detection would facilitate the use of weaner sheep as sentinel animals for the presence of infectious doses of M. a. paratuberculosis on pastures. In controlled pen trials, oral doses of approximately 10(7)-10(8) viable organisms were demonstrated to be infective, whereas doses below 10(4) organisms failed to produce detectable infection. Mycobacterium avium subsp. paratuberculosis (M. a. paratuberculosis) was isolated from intestinal and/or lymphoid tissues collected at necropsy 7 or 14 weeks after first infection, but there were no associated gross or microscopic lesions. Skin testing with intradermal Johnin detected all three infected lambs at 13 weeks post-infection, and one of the three infected lambs at 6 weeks post-infection, with 100% specificity. Results for whole blood IFN-gamma assay showed some correlation with infection status but lacked specificity. One infected lamb gave a positive result in an ELISA for antibodies to M. a. paratuberculosis, 14 weeks post-infection and 1 week after skin testing. This was the first demonstration of experimental infection with S strain M. a. paratuberculosis in Australian Merino sheep at doses likely to be representative of natural infection. Culture from tissues in the first few months post-exposure could facilitate the use of naive weaner sheep as tracer animals to detect heavy contamination of pastures with M. a. paratuberculosis, but low-level contamination may not be detected in such a system.     

A MUCOSAL DISEASE VIRUS AS A CAUSE OF ABORTION, HAIRY BIRTH COAT AND UNTHRIFTINESS IN SHEEP

A condition in Australian sheep resembling border disease was transmitted by the inoculation of pregnant ewes with material from affected lambs. This material contained mucosal disease virus (MDV). Twenty-two lambs comprising 6 from uninoculated control ewes, together with 11 with hairy coats and 5 with normal coats from inoculated ewes, were observed from 7 to 182 days after birth. Nine of the lambs from inoculated ewes died during the experiment from a variety of causes. Glial cell abnormalities were observed in control and affected lambs, but only 4 of the 11 hairy lambs were judged to have abnormal glial cells. There were no consistent histopathological findings indicative of MDV infection. MDV was recovered from tissues of all 11 hairy lambs, but not from any of the lambs with normal coats. The hairy lambs appeared to be immunologically tolerant to the virus. Susceptible sheep in contact with the hairy lambs were infected with MDV. It is suggested that a condition in Australian lambs characterised by hairiness of the birth coat and poor viability is due to foetal infection with a mucosal disease virus.     

Intrauterine and transmammary transmission of Mycobacterium avium subsp paratuberculosis in sheep

OBJECTIVE: To investigate intrauterine infection of foetuses with Mycobacterium avium subsp paratuberculosis and the presence of infection in mammary secretions of sheep. DESIGN: A study of 142 late-pregnant ewes and their foetuses from two heavily infected flocks. PROCEDURE: Infection of ewes was determined at necropsy by histopathology and culture of tissues and mammary secretions. Antemortem tests (clinical assessment, faecal culture and serology) were also applied. Foetuses from 59 infected ewes and 47 apparently uninfected ewes were examined by culture and histopathology. RESULTS: Five of five ewes with clinical ovine Johne's disease had infected foetuses. Only one of 54 subclinically affected ewes, and none of 47 uninfected ewes had an infected foetus. M a paratuberculosis was cultured from mammary secretions or mammary glands of only two of 76 ewes, both of which were clinical cases and had infected foetuses. CONCLUSION: Although intrauterine or transmammary transmission of Mycobacterium avium subsp paratuberculosis may occur frequently in clinically affected sheep, these are less common in subclinically infected ewes. Therefore these modes of transmission are unlikely to compromise existing control programs for ovine Johne's disease on most farms, especially if programs include the immediate culling of clinically affected sheep.     

TRANSMISSION OF A MUCOSAL DISEASE VIRUS INFECTION BETWEEN SHEEP

The transmission of mucosal disease virus (MDV) from infected ewes and their lambs to susceptible sheep was investigated. MDV was recovered from the amniotic fluid of an infected pregnant ewe and from the blood, nasal swabs and urine of hairy lambs. MDV infection was transmitted either at lambing, from infective foetal fluids or lambs, or later as a result of contact with a surviving with a hairy lamb and either aborted or gave birth to an infected hairy lamb. Adult sheep and 12-months-old sheep were less readily infected than were newborn lambs. Pregnant ewes were infected by contact with a hairy lamb and either aborted or gave birth to an infected hairy lamb. A method to minimise spread of the infection in the field is suggested.     

Field trials of a formulation containing moxidectin and 6 in 1 vaccines for sheep

Objective: To assess the efficacy and safety of a formulation containing moxidectin and 6 in 1 vaccine in sheep under field conditions.
Design: Efficacy and safety study.
Animals: Two hundred and five crossbred Merino lambs and two hundred and eight Merino ewes were used in the studies.
Procedure: A formulation was made for the simultaneous treatment of sheep with moxidectin and immunisation against clostridial diseases and caseous lymphadenitis. The efficacy against nematodes, vaccine response and safety were assessed.
Results: Effective control of nematodes and responses to antigens were achieved following subcutaneous administration. The formulation was safe to administer; occasional minor tissue reactions were evident, but no other adverse effects of treatment were observed in either pregnant ewes or lambs, using either the recommended dose, or an overdose of the formulation.
Conclusion: Administration of a formulation containing moxidectin, five clostridial antigens and caseous lymphadenitis antigen proved safe and efficacious under field conditions.