Ramón A. Juste

Prevention strategies against small ruminant lentiviruses: an update.

Small ruminant lentiviruses (SRLVs), including maedi-visna virus (MVV) of sheep and caprine arthritis-encephalitis virus (CAEV), are widespread, cause fatal diseases and are responsible for major production losses in sheep and goats. Seventy years after the legendary maedi-visna sheep epidemic in Iceland, which led to the first isolation of a SRLV and subsequent eradication of the infection, no vaccine or treatment against infection has been fully successful. Research during the last two decades has produced sensitive diagnostic tools, leading to a variety of approaches to control infection. The underlying difficulty is to select the strategies applicable to different epidemiological conditions. This review updates the knowledge on diagnosis, risk of infection, immunisation approaches and criteria for selecting the different strategies to control the spread of SRLVs.

Selection of ovine housekeeping genes for normalisation by real-time RT-PCR; analysis of PrP gene expression and genetic susceptibility to scrapie

BackgroundCellular prion protein expression is essential for the development of transmissible spongiform encephalopathies (TSEs), and in sheep, genetic susceptibility to scrapie has been associated to PrP gene polymorphisms. To test the hypothetical linkage between PrP gene expression and genetic susceptibility, PrP mRNA levels were measured by real-time RT-PCR in six ovine tissues of animals with different genotypes.ResultsPrevious to the PrP gene expression analysis the stability of several housekeeping (HK) genes was assessed in order to select the best ones for relative quantification. The normalisation of gene expression was carried out using a minimum of three HK genes in order to detect small expression differences more accurately than using a single control gene. The expression stability analysis of six HK genes showed a large tissue-associated variation reflecting the existence of tissue-specific factors. Thereby, a specific set of HK genes was required for an accurate normalisation of the PrP gene expression within each tissue. Statistical differences in the normalised PrP mRNA levels were found among the tissues, obtaining the highest expression level in obex, followed by ileum, lymph node, spleen, cerebellum and cerebrum. A tendency towards increased PrP mRNA levels and genetic susceptibility was observed in central nervous system. However, the results did not support the hypothesis that PrP mRNA levels vary between genotypes.ConclusionThe results on PrP gene expression presented here provide valuable baseline data for future studies on scrapie pathogenesis. On the other hand, the results on stability data of several HK genes reported in this study could prove very useful in other gene expression studies carried out in these relevant ovine tissues.

Comparison of different media for the isolation of small ruminant strains of Mycobacterium paratuberculosis.

Two different egg based media, with and without the incorporation of sodium pyruvate, were used to isolate M. paratuberculosis from sheep, goat and cattle samples obtained at our laboratory for two years. Both media were adequate for bovine material, with a slightly improved isolation rate for Herrolds egg yolk medium incorporating sodium pyruvate; however, most of the small ruminant strains grew only on Löwenstein-Jensen medium without sodium pyruvate. Those results point out the need to use different media when working with small ruminant samples and provide further evidence for the existence of different varieties of M. paratuberculosis causing paratuberculosis in cattle and in small ruminants.

Paratuberculosis control: a review with a focus on vaccination.

Mycobacterium avium subsp. paratuberculosis (MAP) infection causes in ruminants a regional chronic enteritis that is increasingly being recognized as a significant problem affecting animal health, farming and the food industry due to the high prevalence of the disease and to recent research data strengthening the link between the pathogen and human inflammatory bowel disease (IBD). Control of the infection through hygiene-management measures and test and culling of positive animals has to date not produced the expected results and thus a new focus on vaccination against this pathogen is necessary. This review summarizes all vaccination studies of cattle, sheep or goats reporting production, epidemiological or pathogenetic effects of vaccination published before January 2010 and that provide data amenable to statistical analyses. The meta analysis run on the selected data, allowed us to conclude that most studies included in this review reported that vaccination against MAP is a valuable tool in reducing microbial contamination risks of this pathogen and reducing or delaying production losses and pathogenetic effects but also that it did not fully prevent infection. However, the majority of MAP vaccines were very similar and rudimentary and thus there is room for improvement in vaccine types and formulations.

Protection against Tuberculosis in Eurasian Wild Boar Vaccinated with Heat-Inactivated Mycobacterium bovis

Tuberculosis (TB) caused by Mycobacterium bovis and closely related members of the Mycobacterium tuberculosis complex continues to affect humans and animals worldwide and its control requires vaccination of wildlife reservoir species such as Eurasian wild boar (Sus scrofa). Vaccination efforts for TB control in wildlife have been based primarily on oral live BCG formulations. However, this is the first report of the use of oral inactivated vaccines for controlling TB in wildlife. In this study, four groups of 5 wild boar each were vaccinated with inactivated M. bovis by the oral and intramuscular routes, vaccinated with oral BCG or left unvaccinated as controls. All groups were later challenged with a field strain of M. bovis. The results of the IFN-gamma response, serum antibody levels, M. bovis culture, TB lesion scores, and the expression of C3 and MUT genes were compared between these four groups. The results suggested that vaccination with heat-inactivated M. bovis or BCG protect wild boar from TB. These results also encouraged testing combinations of BCG and inactivated M. bovis to vaccinate wild boar against TB. Vaccine formulations using heat-inactivated M. bovis for TB control in wildlife would have the advantage of being environmentally safe and more stable under field conditions when compared to live BCG vaccines. The antibody response and MUT expression levels can help differentiating between vaccinated and infected wild boar and as correlates of protective response in vaccinated animals. These results suggest that vaccine studies in free-living wild boar are now possible to reveal the full potential of protecting against TB using oral M. bovis inactivated and BCG vaccines.

A survey of food-borne pathogens in free-range poultry farms

A survey of the occurrence of Campylobacter, Salmonella, Listeria and Shiga toxin-producing E. coli was performed on 60 flocks of free-range chicken from 34 farms in the Basque Country (Northern Spain). Campylobacter was the most prevalent of the four pathogens, isolated in 70.6% of the farms, followed by L. monocytogenes (26.5%), and Salmonella (2.9%). No E. coli O157 or other STEC were isolated. In total 48 flocks from 26 farms were positive for at least one pathogen: 31 of them for a single pathogen (64.6%), and 17 for more than one species (35.4%). C. coli was more prevalent than C. jejuni (15 vs. 13 farms), and both species of Campylobacter were found in 3 farms. L. monocytogenes isolates were identified as serotype 4b complex, and the only Salmonella isolated was serovar Enteritidis. flaA PCR-RFLP performed on 91 Campylobacter isolates (36 C. jejuni and 55 C. coli) yielded 26 patterns, with higher diversity among the C. jejuni isolates. More than one pattern was found in 11 farms, and in 8 of them several patterns were found within the same flock. The findings of the present study suggest that the free-range rearing conditions described herein might have an advantageous effect on diminishing Salmonella but not on Campylobacter or L. monocytogenes flock contamination.

Transmission and control implications of seroconversion to Maedi-Visna virus in Basque dairy-sheep flocks

A retrospective analysis of seroconversion to Maedi-Visna virus (MVV) was carried out for 10 infected semi-intensively reared dairy-sheep flocks that were tested annually between 1994 and 1999. Four of the flocks raised replacement lambs artificially with bovine colostrum and milk replacement to avoid lactogenic MVV infection but did not prevent aerosol contact between replacements and other sheep in the flock. Flock culling percentages ranged between 14 and 25% and in eight flocks the number of sheep that seroconverted was similar to or lower than the number of sheep culled--suggesting that incidence could be reduced by culling seropositive sheep without increasing average culling percentages. Random-effects logistic regression indicated that seroconversion was associated positively with increasing contact with infected sheep and with lifetime MV-serological status of the dam (used as a proxy measure of genetic susceptibility), but not with mode of rearing pre-weaning (artificially or with a seropositive or seronegative dam). Our results indicate that when conditions allow efficient horizontal transmission, there is no evidence that lactogenic infection increases the risk of MV infection and that there is an important inheritable component of disease resistance or susceptibility.

First data on Eurasian wild boar response to oral immunization with BCG and challenge with a Mycobacterium bovis field strain

The Eurasian wild boar (Sus scrofa) is considered a reservoir for bovine tuberculosis (bTB) caused by Mycobacterium bovis and closely related members of the Mycobacterium tuberculosis complex in south-central Spain. The vaccination of wildlife with BCG offers an alternative to culling and to movement restriction for the control of bTB among wildlife reservoirs. In this study, we hypothesized that oral BCG immunization of wild boar would affect the expression of immunoregulatory genes and confer protection against M. bovis. Three groups were used to describe the infection, pathological findings and gene expression profiles in wild boar: BCG-vaccinated and M. bovis-challenged (vaccinated challenged group; N=6), non-vaccinated and M. bovis-challenged (non-vaccinated challenged group; N=4), and non-vaccinated and mock-infected (control group; N=2) animals. M. bovis was isolated from 50% (3/6) and 75% (3/4) of vaccinated challenged and non-vaccinated challenged animals, respectively. All four wild boar from the non-vaccinated challenged group developed bTB-compatible lesions 114 days after challenge. In contrast, only 50% of vaccinated challenged wild boar developed lesions. The PBMC mRNA levels of IL4, RANTES, C3, IFN-gamma and methylmalonyl-CoA mutase (MUT) were analyzed at several days post-vaccination (dpi). When vaccinated challenged animals were compared to controls, all five genes were significantly upregulated at the time of M. bovis infection at 186dpi but IFN-gamma levels were also upregulated at 11 and 46dpi. The C3 and MUT mRNA levels were higher at 46dpi, and 11 and 186dpi, respectively, in vaccinated protected wild boar when compared to non-vaccinated challenged animals. At the end of the experiment (300dpi), the mRNA levels of selected genes were lower in non-vaccinated challenged animals when compared to control wild boar. Exposing wild boar to a dose of 10(4)cfu of M. bovis by the oropharyngeal route is an adequate protocol to produce an infection model in this species. Our results suggested that oral BCG immunization of wild boar results in the upregulation of immunoregulatory genes that may be associated with protective response to M. bovis infection in this species. More studies on vaccine efficacy, delivery, and safety will be needed to confirm if oral vaccination with BCG could be used in bTB control programs for reducing M. bovis infection and clinical disease in wild boar.

Short communication : Investigation of Coxiella burnetii occurrence in dairy sheep flocks by bulk-tank milk analysis and antibody level determination

To estimate the prevalence of Coxiella burnetii in the dairy sheep population from the Basque Country (northern Spain), a study was carried out combining molecular and serological techniques. First, bulk-tank milk samples from 154 flocks belonging to the Latxa Breed Farmers Association were analyzed by PCR, with 22% of flocks testing positive for C. burnetii. Then, a selection of 34 flocks (7 PCR positive and 17 negative) was investigated for the presence of serum antibodies by ELISA test on 1,011 ewes (approximately 30 ewes per flock). A total of 8.9% of the animals were seropositive, 67.6% of the flocks had at least one seropositive animal, but only in 14.7% of them was seroprevalence greater than 25%. Older ewes showed a significantly greater prevalence (17.5%) compared with yearlings (7.5%) or replacement lambs (1.5%). A marginally significant association was found between seroprevalence and PCR detection of C. burnetii in bulk-tank milk. The widespread distribution of C. burnetii in the region advocates for the implementation of Q fever control strategies and highlights the potential risk of sheep as a reservoir and infection source for other domestic and wildlife species and the human population.

Use of a PCR method on fecal samples for diagnosis of sheep paratuberculosis

The high sensitivity of PCR compared to the difficulties of fecal culture in sheep prompted the development of PCR protocols for detection of Mycobacterium avium subsp. paratuberculosis DNA in sheep feces. Although the PCR itself is well developed, and does not pose large technical problems, concentrating the bacteria from samples that may contain low numbers of bacilli using practical methods is still the main difficulty for the use of this technique. In this study, we describe an extraction protocol for the concentration and purification of M. avium subsp. paratuberculosis DNA from fecal samples and we compare it with other methods. The diagnostic performance of the freeze-boiling method was evaluated using a reference method [Vet. Rec. 134 (1994) 95] on fecal samples from a group of selected sheep from different flocks of known individual serological, pathological, and cultural paratuberculosis status. Using, as a reference, a combination of results in those conventional methods, the freeze-boiling PCR protocol showed a sensitivity of 94.1%, and a specificity of 92.3%.