Spatial variation in direct and indirect contact rates at the wildlife-livestock interface for informing disease management.

Little is known about disease transmission relevant contact rates at the wildlife-livestock interface and the factors shaping them. Indirect contact via shared resources is thought to be important but remains unquantified in most systems, making it challenging to evaluate the impact of livestock management practices on contact networks. Free-ranging wild pigs (Sus scrofa) in North America are an invasive, socially-structured species with an expanding distribution that pose a threat to livestock health given their potential to transmit numerous livestock diseases, such as pseudorabies, brucellosis, trichinellosis, and echinococcosis, among many others. Our objective in this study was to quantify the spatial variations in direct and indirect contact rates among wild pigs and cattle on a commercial cow-calf operation in Florida, USA. Using GPS data from 20 wild pigs and 11 cattle and a continuous-time movement model, we extracted three types of spatial contacts between wild pigs and cattle, including direct contact, indirect contact in the pastoral environment (unknown naturally occurring resources), and indirect contact via anthropogenic cattle resources (feed supplements and water supply troughs). We examined the effects of sex, spatial proximity, and cattle supplement availability on contact rates at the species level and characterized wild pig usage of cattle supplements. Our results suggested daily pig-cattle direct contacts occurred only occasionally, while a significant number of pig-cattle indirect contacts occurred via natural resources distributed heterogeneously across the landscape. At cattle supplements, more indirect contacts occurred at liquid molasses than water troughs or molasses-mineral block tubs due to higher visitation rates by wild pigs. Our results can be directly used for parameterizing epidemiological models to inform risk assessment and optimal control strategies for controlling transmission of shared diseases.

Assessment of Babesia bovis 6cys A and 6cys B as components of transmission blocking vaccines for babesiosis.

BACKGROUND: Babesia bovis reproduces sexually in the gut of its tick vector Rhipicephalus microplus, which involves expression of 6cys A and 6cys B proteins. Members of the widely conserved 6cys superfamily are candidates for transmission blocking vaccines (TBV), but intricacies in the immunogenicity of the 6cys proteins in the related Plasmodium parasites required the identification of transmission blocking domains in these molecules for vaccine design. Hereby, the immunogenic efficacy of recombinant (r) B. bovis 6cys A and B proteins as a TBV formulation was studied. METHODS: The immunogenicity of r6cys A and 6cys B proteins expressed in a eukaryotic system was evaluated in a cattle immunization trial (3 immunized and 3 control calves). A B. bovis sexual stage induction in vitro inhibition assay to assess the ability of antibodies to block the production of sexual forms by the parasite was developed. RESULTS: Immunized cattle generated antibodies against r6cys A and r6cys B that were unable to block sexual reproduction of the parasite in ticks. Additionally, these antibodies also failed in recognizing native 6cys A and 6cys B and peptides representing 6cys A and 6cys B functional domains and in inhibiting the development of sexual forms in an in vitro induction system. In contrast, rabbit antibodies generated against synthetic peptides representing predicted B-cell epitopes of 6cys A and 6cys B recognized recombinant and native forms of both 6cys proteins as well as peptides representing 6cys A and 6cys B functional domains and were able to neutralize development of sexual forms of the parasite in vitro. CONCLUSIONS: These data, combined with similar work performed on Plasmodium 6cys proteins, indicate that an effective 6cys protein-based TBV against B. bovis will require identifying and targeting selected regions of proteins containing epitopes able to reduce transmission.

Mechanical transfer of Theileria orientalis: possible roles of biting arthropods, colostrum and husbandry practices in disease transmission.

BACKGROUND: The intracellular protozoal parasite Theileria orientalis has rapidly spread across South-eastern Australia, substantially impacting local cattle industries since 2006. Haemaphysalis longicornis appears to be a biological vector in the endemic regions. Mechanical transfer of blood by biting arthropods, in colostrum or iatrogenic transmission though husbandry procedures is another possible mode of transmission. This study assesses the risk of these mechanical modes of transmission. METHODS: Blood was collected from a T. orientalis Ikeda positive Angus steer, and was inoculated into the jugular vein of 9 calves in 3 treatment groups, each with 3 animals. Calves in Group 1 received 10 ml of cryopreserved blood, while those in Groups 2 and 3 received 1 ml (fresh blood) and 0.1 ml (cryopreserved), respectively. An additional three animals remained as negative controls and the donor calf was also followed as a positive control. Blood was collected over 3 months, and analysed via qPCR for the presence of the parasite. Samples of the sucking louse Linognathus vituli were collected opportunistically from calves 5 months after inoculation and tested for T. orientalis. For the colostral transmission study, 30 samples of blood and colostrum were collected from cows at calving in an endemic herd. These samples along with blood from their calves were tested by qPCR for T. orientalis and for antibodies to the major piroplasm surface protein (MPSP). RESULTS: Eight of the nine inoculated calves became positive for T. orientalis. The prepatent period of these infections was inversely correlated with inoculation dose. All negative control calves remained negative and the positive control calf remained positive. Samples of L. vituli tested positive for T. orientalis Ikeda, while some samples of colostrum were also shown to be qPCR and anti-MPSP positive. All calves in the colostral study tested qPCR negative although one was antibody-positive. CONCLUSIONS: T. orientalis is capable of being mechanically transferred by intravenous inoculation with small volumes of blood and is detectable up to 5 months post-infection. Animals infected by this means may play a significant role in the transmission of the disease by acting as asymptomatic carriers. Other modes of blood transfer, including biting arthropods and colostral transfer are also possible modes of disease transmission.

Dam Mycobacterium avium subspecies paratuberculosis (MAP) infection status does not predetermine calves for future shedding when raised in a contaminated environment: a cohort study.

Uptake of Mycobacterium avium subsp. paratuberculosis (MAP) by calves in the first days of life from colostrum, milk and faeces is regarded an important moment of transmission. The objective of this study was to quantify the association between the MAP status of dams as determined by the presence of MAP DNA and antibody in colostrum and that of DNA in faeces and the environment with subsequent MAP shedding of their daughters. A cohort of 117 dam-daughter pairs giving birth/being born on eight commercial dairy farms with endemic paratuberculosis was followed where colostrum, faecal and environmental samples (dust) were analysed for the presence of MAP using an IS900 real-time PCR. Antibodies in colostrum were measured by ELISA. Analysis of dust samples showed that on all farms environmental MAP exposure occurred continuously. In significantly more colostrum samples (48%) MAP DNA was detected compared to faecal samples (37%). MAP specific antibodies were present in 34% of the colostrum samples. In total MAP DNA was present in faecal samples of 41% of the daughters at least once during the sampling period. The association between faecal shedding in the offspring and the dam MAP status defined by MAP PCR on colostrum, MAP PCR on faeces or ELISA on colostrum was determined by an exact cox regression analysis for discrete data. The model indicated that the hazard for faecal shedding in daughters born to MAP positive dams was not significantly different compared to daughters born to MAP negative dams. When born to a dam with DNA positive faeces the HR was 1.05 (CI 0.6; 1.8) and with DNA positive colostrum the HR was 1.17 (CI 0.6; 2.3). When dam status was defined by a combination of both PCR outcomes (faeces and colostrum) and the ELISA outcome the HR was 1.26 (CI 0.9; 1.9). Therefore, this study indicates that neither the presence of MAP DNA in colostrum, MAP DNA in faeces nor the presence of MAP antibodies in colostrum of the dam significantly influences the hazard of MAP shedding in their subsequent daughters up to the age of two years when raised in a contaminated environment.

What attracts elk onto cattle pasture? Implications for inter-species disease transmission.

In Southwest Alberta, beef cattle and wild elk (Cervus elaphus) have similar habitat preferences. Understanding their inter-species contact structure is important for assessing the risk of pathogen transmission between them. These spatio-temporal patterns of interactions are shaped, in part, by range management and environmental factors affecting elk distribution. In this study, resource selection modeling was used to identify factors influencing elk presence on cattle pasture and elk selection of foraging patches; furthermore, consequences for inter-species disease transmission were discussed. Data on pasture management practices and observations of elk were collected from 15 ranchers during interviews. Pasture use by elk was defined based on telemetry data (from GPS collars deployed on 168 elk in 7 herds) and rancher observations. At the patch scale, foraging patches used by elk were identified by spatio-temporal cluster analysis of telemetry data, whereas available patches were randomly generated outside the area delimited by used patches. For pastures and patches, landscape and human-managed features were characterized using remote sensing data and interviews, respectively. Attributes of available and used pastures (or patches) were compared using resource selection functions, on annual and seasonal (or annual and monthly) time scales. Additionally, intensity of pasture use was modeled using negative binomial regression. Cultivated hay land and mineral supplements were associated with elk presence on cattle pastures, whereas pastures with manure fertilization and higher traffic-weighted road densities were less likely to be used by elk. The effects of landscape (elevation, aspect, water access) and vegetation (forest cover, Normalized Difference Vegetation Index) characteristics on patch selection were consistent with typical elk habitat requirements. The presence of cattle and the traffic-weighted road density were negatively associated with patch selection. The apparent avoidance of cattle by elk reduced the risk of direct transmission of pathogens, except during winter months. However, human-managed features attracting elk to cattle pastures (e.g. hay land and mineral supplements) may increase inter-species pathogen transmission through indirect contacts.

Effect of freezing treatment on colostrum to prevent the transmission of bovine leukemia virus.

Here, we used a sheep bioassay to determine the effect of freezing colostrum to prevent the transmission of bovine leukemia virus (BLV) among neonatal calves. Leukocytes were isolated from the colostrum of a BLV-infected Holstein cow and were then either left untreated (control) or freeze-thawed. A sheep inoculated intraperitoneally with the untreated leukocytes was infected with BLV at 3 weeks after inoculation, whereas the sheep inoculated with treated leukocytes did not become infected. The uninfected sheep was inoculated again with leukocytes isolated from the colostrum of another BLV-infected Holstein cow after freezing treatment, and again it did not become infected with BLV. Finally, this sheep was inoculated with the leukocytes isolated from the colostrum of another virus-infected cow without freezing treatment, and it became infected with BLV at 4 weeks after inoculation. The results indicate that colostrum should be frozen as a useful means of inactivating the infectivity of BLV-infected lymphocytes.

Implementing a probabilistic definition of freedom from infection to facilitate trade of livestock: putting theory into praxis for the example of bovine herpes virus-1.

International trade of livestock and livestock products poses a significant potential threat for spread of diseases, and importing countries therefore often require that imported animals and products are free from certain pathogens. However, absolute freedom from infection cannot be documented, since all test protocols are imperfect and can lead to false-negative results. It is possible instead to estimate the "probability of freedom from infection" and its opposite, the probability of infection despite having a negative test result. These probabilities can be estimated based on a pre-defined target prevalence, known surveillance efforts in the target population and known test characteristics of any pre-export test. Here, calculations are demonstrated using the example of bovine herpes virus-1 (BoHV-1). In a population that recently became free of BoHV-1 without using vaccination, the probability of being infected of an animal randomly selected for trade is 800 per 1 million and this probability is reduced to 64 (95% probability interval PI 6-161) per 1 million when this animal is tested negatively prior to export with a gB-ELISA. In a population that recently became free of BoHV-1 using vaccination, the probability of being infected of an animal randomly selected for trade is 200 per 1 million, and this probability can be reduced to 63 (95% PI 42-87) when this animal is tested negatively prior to export with a gE-ELISA. Similar estimations can be made on a herd level when assumptions are made about the herd size and the intensity of the surveillance efforts. Subsequently, the overall probability for an importing country of importing at least 1 infected animal can be assessed by taking into account the trade volume. Definition of the acceptable level of risk, including the probability of false-negative results to occur, is part of risk management. Internationally harmonized target prevalence levels for the declaration of freedom from infection from selected pathogens provide a significant contribution to the facilitation of international trade of livestock and livestock products by allowing exporting countries to design tailor-made output-based surveillance programs, while providing equivalent guarantees regarding the probability of freedom from infection of the population. Combining this with an approach to assess the overall probability of introducing at least 1 infected animal into an importing country during a defined time interval will help importing countries to achieve their desired level of acceptable risk and will help to assess the equivalence of animal health and food safety standards between trading partners.

Evaluation of the risk of paratuberculosis in adult cows fed Mycobacterium avium subsp paratuberculosis DNA-positive or -negative colostrum as calves.

OBJECTIVE: To estimate the risk of subclinical Mycobacterium avium subsp paratuberculosis (MAP) infection in cows that ingested MAP DNA-positive raw colostrum as calves, compared with risk in cows that ingested MAP DNA-negative raw colostrum as calves. ANIMALS: 205 calves born in 12 commercial dairy herds. PROCEDURES: Each calf was separated from its dam within 30 to 60 minutes after birth and fed raw colostrum. For each calf, samples of the colostrum fed were collected and tested for the presence of MAP DNA by use of a nested PCR assay for the target gene ISMAP02. Calves fed colostrum positive or negative for MAP DNA were classified into exposed (n = 69) and unexposed (136) groups, respectively. Each calf was tested for MAP infection at 30, 42, and 54 months of age by use of a serum ELISA and bacterial culture of feces. Weibull hazard regression models were used to evaluate the association between exposure to MAP DNA-positive colostrum and time to testing positive for MAP infection. RESULTS: Hazard of MAP infection was not different between groups (exposed vs unexposed) when serum ELISA, bacterial culture of feces, or both diagnostic tests (parallel interpretation) were positive. CONCLUSIONS AND CLINICAL RELEVANCE: Heifer calves fed MAP DNA-positive colostrum were at no greater risk of MAP infection, compared with heifer calves fed MAP DNA-negative colostrum. This result contradicts findings from other studies and should be interpreted with caution.

[Reproducibility of bovine neonatal pancytopenia (BNP) via the application of colostrum]. / Reproduzierbarkeit der bovinen neonatalen Panzytopenie (BNP) über die Verabreichung von Kolostrum.

A haemorrhagic diathesis has been observed in young calves since 2007 which is described as bovine neonatal pancytopenia (BNP) and presents a completely new disease. The objectives of our investigation were to test if BNP could be reproduced using colostrum of cows with a BNP history and pre-colostral calves from farms where BNP has not been observed. In the present experiment, 22 German Holstein calves from BNP-free farms were fed four to six hours after birth 2.5 l colostrum from cows which had been reported to have had at least one calf with BNP in the last lactation. We distinguished three different experimental groups according to the composition of the colostrum. In experimental group I, each of the six calves received colostrum of a single cow, in experimental group II all six calves received colostrum from the same cow and in experimental group III each of the ten calves received a colostrum mix from ten different cows. Clinical signs of BNP were observed in 50% of the calves in experimental group I, 67% of the calves in experimental group II and all calves in experimental group III. The lethality in the three experimental groups was significantly different with rates of 16.7%, 66.7% and 80%, respectively. Calves fed with a colostrum-mix in experimental group III had the highest lethality. Neither the farm nor the amount of the colostrum fed had a significant effect on the occurrence and course of BNP. The profiles for thrombocytes, leucocytes and erythrocytes significantly differed in dependence of the severity of BNP signs. Calves with non-lethal BNP showed thrombocytopenia with values below 100 G/l on the 1th to 3rd and the 7th to 11th day of life. In calves with lethal BNP, thrombocytes decreased under 50 G/l from day 5. In calves with non-lethal BNP, a decrease of the leucocytes under the threshold was present only for a short period of time. In calves with lethal BNP, leucocytes decreased in the first 5 days after birth continuously and increased on the 6th to the 8th day to normal values and then a rapid decrease occurred. Erythrocytes decreased under the normal threshold just in the last two days before the calves died or were euthanized. Thus, the present experiments showed that colostrum of cows with a BNP-history and vaccination with PregSure BVD from Pfizer caused lethal BNP. We can assume that the different reactions of the calves are due to immunogenetic reactions to colostral alloreactive antibodies. The reaction spectrum of calves depends on the presence of antigens which can react with these colostral antibodies. The experimental results can explain the different incidences of BNP within and among farms as well as between breeds.

Predicting fadeout versus persistence of paratuberculosis in a dairy cattle herd for management and control purposes: a modelling study.

Epidemiological models enable to better understand the dynamics of infectious diseases and to assess ex-ante control strategies. For Mycobacterium avium subsp. paratuberculosis (Map), possible transmission routes have been described, but Map spread in a herd and the relative importance of the routes are currently insufficiently understood to prioritize control measures. We aim to predict early after Map introduction in a dairy cattle herd whether infection is likely to fade out or persist, when no control measures are implemented, using a modelling approach. Both vertical transmission and horizontal transmission via the ingestion of colostrum, milk, or faeces present in the contaminated environment were modelled. Calf-to-calf indirect transmission was possible. Six health states were represented: susceptible, transiently infectious, latently infected, subclinically infected, clinically affected, and resistant. The model was partially validated by comparing the simulated prevalence with field data. Housing facilities and contacts between animals were specifically considered for calves and heifers. After the introduction of one infected animal in a naive herd, fadeout occurred in 66% of the runs. When Map persisted, the prevalence of infected animals increased to 88% in 25 years. The two main transmission routes were via the farm's environment and in utero transmission. Calf-to-calf transmission was minor. Fadeout versus Map persistence could be differentiated with the number of clinically affected animals, which was rarely above one when fadeout occurred. Therefore, early detection of affected animals is crucial in preventing Map persistence in dairy herds.

Use of ethnoveterinary remedies in the management of foot and mouth disease lesions in a diary herd.

An outbreak of Foot and Mouth Disease (FMD) affecting 95 (57.2%) out of 166 cattle occurred in a medium-scale dairy farm in Kikuyu district, Kenya. Ethnoveterinary remedies of natural Soda ash solution (97% sodium bicarbonate), honey and finger millet flour were used to manage the FMD lesions. The lesions were washed with soda ash solution to remove the necrotic tissue after which raw honey and finger millet flour were applied to the cleaned lesions. The lesions were examined daily and those with necrotic material washed again with the Soda ash solution. Honey and finger millet flour were applied daily for three days. There was rapid healing of the lesions with the animals resuming feeding after three days. The fast healing of the lesions vindicates the use of these cheap, locally available and easy to apply products in the management of FMD lesions. However, more studies are needed to evaluate further their potencies.

Perineal swabs reveal effect of super shedders on the transmission of Escherichia coli O157:H7 in commercial feedlots.

Cattle that shed more than 10(4) cfu/g of Escherichia coli O157 in feces have been described as super shedders (SS) and are thought to have major impacts on prevalence and transmission of this organism. Two Southern Alberta commercial feedlots (feedlot X, 7 pens averaging 183 steers; feedlot Y, 5 pens averaging 153 steers) were sampled from May 2007 to January 2008. Background samples [fecal pat (FP) water, ropes] were taken weekly from each pen for 2 wk before collection of samples from individuals [fecal grab (FG); perineal swab] at 2 different times [during spring and summer (S1); immediately before slaughter during fall and winter (S2)]. Immunomagnetic separation and selective media were used for detecting E. coli O157:H7. Positive FG and FP were enumerated by direct plating onto sorbitol MacConkey agar supplemented with 2.5 mg/L of potassium tellurite and 0.05 mg/L of cefixime. Five sorbitol-negative colonies were agglutinated using an anti-O157 latex kit, and the proportion of positive colonies was adjusted for non-E. coli O157:H7. Overall, there were 153 (7.16%) and 10 (0.45%) SS at S1 and S2, respectively. In feedlot X, SS and penmates of SS during S1 were more likely (P < 0.01) to shed E. coli O157:H7 in their feces and have this organism on their perineum than cattle in a pen where no SS were identified. In feedlot Y, SS and penmates of SS during S1 were more likely (P < 0.01) to have E. coli O157:H7 on their perineum than those from a pen where only 1 SS was identified, but steers in only 1 pen with multiple SS were more likely (P < 0.01) to shed this organism in feces. Overall, E. coli O157:H7 was 1.85 times more likely (P < 0.01) to be detected in perineal swabs compared with FG and E. coli O157:H7 was more likely (P < 0.01) to be detected at S1 compared with S2 for all sample types. Super shedders were a larger proportion of shedding cattle in S1 than in S2, but the presence of SS increased (P < 0.01) prevalence of this organism on the perineum of cattle throughout the year. Even when SS did not increase fecal shedding of E. coli O157:H7, their presence increased contamination of hides, an outcome that could have important implications for contamination of carcasses at the abattoir.

Efficacy of feeding plasma-derived commercial colostrum replacer for the prevention of transmission of Mycobacterium avium subsp paratuberculosis in Holstein calves.

OBJECTIVE: To estimate the relative risk of paratuberculosis (Johne's disease [JD]) in calves fed a plasma-derived colostrum-replacement (CR) product versus raw bovine maternal colostrum (MC). STUDY DESIGN: Randomized controlled clinical trial. ANIMALS: 497 heifer calves born in 12 JD-endemic commercial Holstein dairy farms located in Minnesota and Wisconsin. PROCEDURES: Every calf was separated from its dam within 30 to 60 minutes after birth and systematically assigned to be fed raw bovine MC (control group, n = 261 calves) or CR (treatment group, 236 calves). The calves were monitored to adulthood and tested for Mycobacterium avium subsp paratuberculosis (MAP) infection by use of an ELISA to detect serum antibodies against MAP and bacterial culture for MAP in feces at approximately 30, 42, and 54 months of age. Weibull regression models were used to evaluate the effect of feeding CR (vs raw bovine MC) on the risk of developing JD infection. RESULTS: Calves fed CR at birth were less likely (hazard ratio = 0.559) to become infected with MAP (as determined by use of an ELISA, bacterial culture, or both diagnostic tests), compared with the likelihood for calves fed MC at birth. CONCLUSIONS AND CLINICAL RELEVANCE: This study revealed that feeding CR reduced the risk of developing MAP infection in Holstein calves born in JD-endemic herds, which implied that feeding raw bovine MC may be a source of MAP for calves. Plasma colostrum-replacement products may be an effective management tool for use in dairy herds attempting to reduce the prevalence of JD.

Oral and rectal administration of bacteriophages for control of Escherichia coli O157:H7 in feedlot cattle.

This study compared oral and rectal administration of O157-specific bacteriophages for mitigating the fecal shedding of Escherichia coli O157 by experimentally inoculated steers. Fecal shedding of nalidixic acid-resistant (Nal(R)) E. coli O157:H7 was monitored over 83 days after oral (ORL; 3.3 x 10(11) PFU), rectal (REC; 1.5 x 10(11) PFU), both oral and rectal (O+R; 4.8 x 10(11) PFU), or no (CON; control) treatment with a four-strain O157-specific bacteriophage cocktail in multiple doses. Bacteriophages were enumerated by plaque assay, and NalR E. coli O157:H7 by direct plating on sorbitol MacConkey agar supplemented with cefixime, potassium tellurite, and nalidixic acid. Orally treated steers produced the fewest Nal(R) E. coli O157:H7 culture-positive samples (P < 0.06) compared with REC and O+R steers, but this number was only nominally lower (P = 0.26) than that for the CON steers. The overall mean shedding level (log CFU per gram of feces) was higher for REC steers (P < 0.10) than for steers of the other treatment groups. Despite the shedding of higher mean bacteriophage levels (log PFU per gram of feces) by ORL and O+R than by CON and REC steers, there was no difference (P > 0.05) in the number of E. coli O157-positive samples among treatments. Bacteriophage was isolated from CON steers, indicating that these steers acquired the bacteriophage from the environment and shed the phage at a level similar to that of REC steers (P = 0.39). Continuous bacteriophage therapy may be an efficacious method for mitigating shedding of E. coli O157:H7 in cattle, providing that the host bacterium does not develop resistance. This therapy may be especially advantageous if nontreated cattle can acquire this biocontrol agent from the feedlot environment.

Colostrum and milk as risk factors for infection with Mycobacterium avium subspecies paratuberculosis in dairy cattle.

Mycobacterium avium ssp. paratuberculosis (MAP) infections cause major losses to the dairy industry. Transmission of MAP occurs primarily via feces and in utero, but MAP can also be excreted in colostrum and milk. The objective of this study was to determine whether colostrum and milk fed to calves are important risk factors for infection with MAP. A questionnaire was sent to 1,050 farms participating in the Danish control program on paratuberculosis in early 2007. Details on practices regarding colostrum and milk feeding between 1999 and 2006 were obtained from 808 (77%) herds. Nine vaccinated herds were excluded. Information on MAP antibody-ELISA results, date of birth, and herd of birth of 93,994 animals was obtained from the Danish Cattle Database. A 2-level logistic regression model was fitted with a dichotomized ELISA response, with outcome, age, source of colostrum, and milk as fixed effects, and herd as a random effect. Animals fed colostrum from multiple cows had an odds ratio of 1.24 of being ELISA positive compared with animals fed colostrum from their own dam only. Calves suckling with foster cows had an odds ratio of 2.01 of being ELISA positive compared with calves fed milk replacer. Feeding bulk tank milk and pooled milk from cows with high somatic cell counts did not increase the risk of being ELISA positive. Overall, the results of the study suggested that source of milk was not of great importance for the transmission of MAP, but colostrum should be fed only from the dam of that calf.

Pasteurization of colostrum reduces the incidence of paratuberculosis in neonatal dairy calves.

In the present study, the potential benefits of feeding pasteurized colostrum were demonstrated in calves born to dams naturally infected with Mycobacterium avium ssp. paratuberculosis. Calves were separated at birth from their dams and randomly allocated into a group fed either the colostrum of their dam (DC; n = 6), followed by feeding the milk of the dam for 3 wk and then milk replacer, or into a group fed pooled pasteurized colostrum (PC; n = 5) from healthy noninfected dams, followed by milk replacer. At 6 wk of age, calves were weaned onto calf starter, housed together, and fed in a similar manner throughout the rest of the 12-mo study. Calves were necropsied at the end of the study, and 25 tissue sites were sampled from each animal and cultured for M. avium ssp. paratuberculosis. Sixteen of the 25 tissue sites were positive for calves across both treatment groups, with 14 of the 16 tissue sites positive for DC calves and 9 of the 16 tissue sites positive for PC calves. The degree of colonization within a tissue was low and variable for calves within treatment groups, and fecal shedding of M. avium ssp. paratuberculosis was minimal during the 12-mo study. As a measure of the early immune response to infection, blood obtained from calves was stimulated in vitro with M. avium ssp. paratuberculosis antigen preparations, and IFN-Upsilon secretion was measured. Antigen-specific IFN-Upsilon was consistently greater throughout the study in DC calves (0.95 +/- 0.19) compared with PC calves (0.43 +/- 0.10). Although long-term benefits are unknown, these results indicate that feeding a source of colostrum from paratuberculosis-free dams may decrease the initial exposure of neonates to M. avium ssp. paratuberculosis, perhaps decreasing dissemination of infection over time.

Immune response to Neospora caninum in naturally infected heifers and heifers vaccinated with inactivated antigen during the second trimester of gestation.

The objective of this study was to compare the immune response to Neospora caninum in naturally infected heifers and heifers inoculated with a killed whole N. caninum tachyzoite preparation during the second trimester of gestation. Nine Holstein heifers were used in this study; three naturally infected heifers were born from seropositive dams, and six seronegative heifers were born from seronegative dams. Four seronegative heifers were subcutaneously vaccinated with a killed whole N. caninum tachyzoite preparation at weeks 13, 15 and 17 of gestation. A killed whole N. caninum tachyzoite preparation containing 45 mg of protein/5 ml dose was formulated with 70% of mineral oil adjuvant (13% consisting of Arlacel C, 85% Marcol 52 and 2% Tween-80). Similarly, two seronegative heifers (negative controls) were inoculated with mock-infected bovine monocytes in oil adjuvant. Humoral immune responses were tested by using an indirect fluorescent antibody test (IFAT) and an indirect enzyme-linked immunosorbent assay (ELISA) for detecting isotype specific antibodies. Cellular immune responses were assessed by lymphocyte proliferation test (LPT) and IFN-gamma production. N. caninum-specific antibody responses increased in immunized cattle by week 15 of gestation (mean reciprocal antibody titers 450+/-252), peaked at week 23 (mean 16,000+/-6400). Maximum antibody response in naturally infected heifers was observed at week 19 of gestation (mean: 3467+/-2810). Mean serum IFAT titers were significantly higher in immunized heifers compared with those in naturally infected heifers from weeks 17 to 25 (P < 0.05). Analysis of isotype specific antibodies in naturally infected heifers revealed a predominant IgG1 response in one heifer and a predominant IgG2 response in the other two. Similar titers of IgG1 and IgG2 occurred in immunized heifers. Control heifers remained seronegative throughout the study by IFAT and ELISA. Significant antigen-specific proliferation responses were only detected in naturally infected heifers in week 19 of gestation. Peripheral mononuclear blood cells (PMBC) from immunized animals produced IFN-gamma in similar concentrations to those of infected animals (P > 0.05). No abortion was seen in any experimental group; however, one calf from a vaccinated heifer died due to dystocia. All calves from vaccinated and control heifers were seronegative by IFAT at 6 months of age; in contrast, calves born from naturally infected heifers remained seropositive with titers > or = 200. Killed vaccine induced similar immune responses to those found in chronically, naturally infected cattle which did not abort; however, different immune pathways may be followed in vaccinated and natural infected heifers with differences in degree of protective immunity.

Effect of supplementing corn- or barley-based feedlot diets with canola oil on faecal shedding of Escherichia coli O157:H7 by steers.

AIMS: This study was conducted to evaluate the effect of supplementing barley- or corn-based diets with canola oil on faecal shedding of Escherichia coli O157:H7 by experimentally inoculated feedlot cattle. METHODS AND RESULTS: Four groups of yearling steers fed on barley- or corn-based feedlot diets containing 0% (BA; CO) or 6% canola oil (BA-O; CO-O) were inoculated with 10(10) CFU of a mixture of four nalidixic acid-resistant strains of E. coli O157:H7. The inoculated strains were tracked in oral (mouth swab) and environmental (water, water bowl interface, feed, faecal pat) samples by enrichment and immunomagnetic separation (IMS) for 12 weeks, and in rectally collected faecal samples for 23 weeks (enumeration by dilution plating for 12 weeks; detection by IMS for a further 11 weeks). Levels of E. coli O157:H7 shed in faecal samples over the course of the enumeration period were similar (P = 0.14) among treatments. Disappearance of the inoculated strains from faeces was more rapid (P = 0.009) with barley than with corn, but shedding levels at the end of the enumeration period were similar (P = 0.21) across grain types. Canola oil supplementation did not affect (P = 0.71) the rate of disappearance of E. coli O157:H7 from faeces. The numbers of steers culture positive for E. coli O157:H7 during the enumeration period were similar (P = 0.57) among treatments. During the 11-week detection period, however, more (P < 0.001) steers were E. coli O157:H7-positive in the BA group (15/64) than in BA-O (two of 64), CO (two of 56), or CO-O (one of 56). The organism was present in two of 48 water samples (both CO-O), one of 48 water bowl swabs (BA-O), four of 48 feed samples (two of 12 BA; two of 12 CO-O), 30 of 48 pen floor faecal pat samples, and 296 of 540 mouth swabs (81/144 BA, 80/144 BA-O, 74/126 CO and 61/126 CO-O). CONCLUSION: Supplementing corn or barley-based diets with canola oil did not affect shedding of E. coli O157:H7 by feedlot cattle. SIGNIFICANCE AND IMPACT OF THE STUDY: High-shedding individuals (i.e. 'super shedders') may be responsible for disseminating E. coli O157:H7 among penmates. Faeces on pen floors appears to be a more significant source of infection than are feed or drinking water.

Isolation of Neospora caninum from a calf in Malaysia.

In order to attempt isolate the protozoan parasite Neospora caninum, an N. caninum seropositive pregnant Sahiwal Friesian cross heifer from a large-scale dairy farm in Malaysia was kept for observation until parturition at the Veterinary Research Institute, Ipoh. The heifer gave birth to a female calf that was weak, underweight and unable to rise. Precolostral serum from the calf had an N. caninum indirect fluorescent antibody test titre of 1:3200. It died 12 h after birth and necropsy was performed. Brain homogenate from the calf was inoculated into 10 BALB/c mice that were kept for 3 months after which brain tissue from the mice was inoculated onto 24 h fresh monolayer Vero cell lines. The cell cultures were examined daily until growth of intracellular protozoa was observed. DNA of the organisms from the cell cultures was analyzed by PCR and DNA sequencing. DNA fragments of the expected size were amplified from the isolate using N. caninum-specific primers, and sequence analysis of ITS1 clearly identified the isolate as N. caninum. This is the first successful isolation of N. caninum from a bovine in Malaysia, and the isolate is designated Nc-MalB1.

DNA vaccines against anthrax.

DNA vaccination is vaccination at its simplest. Due to renewed interest in vaccination against anthrax and other biothreat agents, a genetic immunisation approach offers attractive possibilities for rapid, responsive vaccine development. DNA vaccination against anthrax is an active area of research showing promising results at present, which in the short-term and in the future could form the basis for new advances in multi-agent vaccine development. The anthrax 'model' constitutes an important experimental system for genetic immunisation technology development.