Paratuberculosis vaccination specific and non-specific effects on cattle lifespan.

Records of cattle vaccination against paratuberculosis (PTB) have been analyzed to determine whether or not non-specific effect (NSE) on overall mortality similar to that observed in BCG vaccinated humans occurs in animals. The results of a previously reported slaughterhouse study on PTB prevalence were used as a reference on the age incidence of advanced patent (clinical) epidemio-pathogenic forms. In the proper vaccine study, cows in 30 cattle farms in the Basque Country, Spain were followed-up for between 1 and 13 years. Vaccinated groups were composed by 1008 (592 right-censored) animals younger than 3 months treated as calves and by 3761 (3160 right-censored) vaccinated at any older age. Controls were 339 (157 right-censored) and 4592 (2213 right-censored) age matched animals, respectively. Individual last year presence in the annual testing was considered age at culling or death. A survival analysis was carried out according age at vaccination of vaccinated versus non-vaccinated animals. PTB age incidence in the slaughterhouse study was subtracted from the difference between vaccinated and non-vaccinated animals at the same age in order to estimate PTB-specific and non-specific effects. The maximum difference was observed at the 2-3 years interval with a 33.9% mortality reduction in the calf vaccinated group. This corresponded also with the maximum NSE that was 24.5% for a PTB incidence of 9.5%. Overall, vaccination afforded to calves a 26.5% yearly mortality protection, split between 11.1% PTB-specific and 15.4% NSE. These results support a NSE on total mortality associated with PTB vaccination that appeared to persist for up to 6-7 years. This confirms for the first time in an animal field study the innate immune system memory predicted by the recently proposed trained immunity theory. Contrasting the literature, no deleterious effects of killed vaccines on females were observed. Mortality reduction would offset vaccination costs and could improve livestock systems efficiency and potentially reduce antibiotic use. Clinical trial registered with Spanish Agency for Drugs and Sanitary products (AEMPS) as 11/012/ECV.

Clinical course and pathogenicity of variant rabbit haemorrhagic disease virus in experimentally infected adult and kit rabbits: Significance towards control and spread.

RHDVb has become the dominant RHDV on the Iberian Peninsula. A better understanding of its pathogenicity is required to aid control measures. Thus, the clinical course, humoral immune response, viraemia and kinetics of RHDV-N11 (a Spanish RHDVb isolate) infection in different tissues at both viral RNA and protein levels were studied in experimentally infected young and adult rabbits. The case fatality rate differed between the two age groups, with 21% of kits succumbing while no deaths were observed in adults. Fever and viremia were strongly associated with death, which occurred 48 h post infection (PI) too fast for an effective humoral immune response to be mounted. A significant effect on the number of viral RNA copies with regard to the variables age, tissue and time PI (p < 0.0001 in all cases) was detected. Histological lesions in infected rabbits were consistently more frequent and severe in liver and spleen and additionally intestine in kits, these tissues containing the highest levels of viral RNA and protein. Although no adults showed lesions or virus antigen in intestine, both kits and adults maintained steady viral RNA levels from days 1 to 7 PI in this organ. Analysis revealed the fecal route as the main dissemination route of RHDV-N11. Subclinically infected rabbits had detectable viral RNA in their faeces for up to seven days and thus may play an important role spreading the virus. This study allows a better understanding of the transmission of this virus and improvement of the control strategies for this disease.

Immune response profiles of calves following vaccination with live BCG and inactivated Mycobacterium bovis vaccine candidates.

Conventional control and eradication strategies for bovine tuberculosis (BTB) face tremendous difficulties in developing countries; countries with wildlife reservoirs, a complex wildlife-livestock-human interface or a lack of veterinary and veterinary public health surveillance. Vaccination of cattle and other species might in some cases provide the only suitable control strategy for BTB, while in others it may supplement existing test-and-slaughter schemes. However, the use of live BCG has several limitations and the global rise of HIV/AIDS infections has furthermore warranted the exploration of inactivated vaccine preparations. The aim of this study was to compare the immune response profiles in response to parenteral vaccination with live BCG and two inactivated vaccine candidates in cattle. Twenty-four mixed breed calves (Bos taurus) aged 4-6 months, were allocated to one of four groups and vaccinated sub-cutaneously with live M. bovis BCG (Danish 1331), formalin-inactivated M. bovis BCG, heat-killed M. bovis or PBS/Montanide™ (control). Interferon-γ responsiveness and antibody production were measured prior to vaccination and at weekly intervals thereafter for twelve weeks. At nine weeks post-priming, animals were skin tested using tuberculins and MTBC specific protein cocktails and subsequently challenged through intranodular injection of live M. bovis BCG. The animals in the heat-killed M. bovis group demonstrated strong and sustained cell-mediated and humoral immune responses, significantly higher than the control group in response to vaccination, which may indicate a protective immune profile. Animals in this group showed reactivity to the skin test reagents, confirming good vaccine take. Lastly, although not statistically significant, recovery of BCG after challenge was lowest in the heat-killed M. bovis group. In conclusion, the parenteral heat-killed M. bovis vaccine proved to be clearly immunogenic in cattle in the present study, urging further evaluation of the vaccine in challenge studies using virulent M. bovis and assessment of vaccine efficacy in field conditions.

Paratuberculosis in Latin America: a systematic review.

Latin America is the definition of the American group, where languages of Latin origin are spoken, including countries in South, Central, and North America. Paratuberculosis is a gastrointestinal contagious chronic disease that affects ruminants, whose etiological agent is the bacilli Mycobacterium avium subsp. paratuberculosis (MAP). Paratuberculosis is characterized by intermittent diarrhea, decreased milk production, dehydration, and progressive weight loss and is possibly involved in Crohn's disease, a human intestinal disease. MAP is resistant to environmental factors, pasteurization, and water disinfection, which coupled with the subclinical-clinical nature of the disease, and makes paratuberculosis a relevant socioeconomic and public health issue, justifying the descriptive review of research on the disease carried out in Latin American countries. A survey of articles, published until September 2016, on the Scopus database, PubMed, Agris, and Science Direct, about detection of the agent and the disease in Latin America, without restrictions to the date of the research was performed. The keywords were as follows: "paratuberculosis," "Mycobacterium avium subsp. paratuberculosis," "cattle," "milk," "wildlife," "goat," "ovine," "dairy," and the name of each country in English. Studies found from nine of the 20 Latin America countries, 31 related to Brazil, 17 to Argentina, 14 to Chile, eight to Colombia, six to Mexico, two to Peru, two to Venezuela, and one to Panama and to Bolivia, each. The agent was detected in cattle, goats, sheep, domesticated water buffalo, and wild animals. Microbiological culture, PCR, and ELISA were the frequent techniques. The small number of studies may result in overestimation or underestimation of the real scenario.

Vaccination against Louping Ill Virus Protects Goats from Experimental Challenge with Spanish Goat Encephalitis Virus.

Spanish goat encephalitis virus (SGEV) is a recently described member of the genus Flavivirus belonging to the tick-borne encephalitis group of viruses, and is closely related to louping ill virus (LIV). Naturally acquired disease in goats results in severe, acute encephalitis and 100% mortality. Eighteen goats were challenged subcutaneously with SGEV; nine were vaccinated previously against LIV and nine were not. None of the vaccinated goats showed any clinical signs of disease or histological lesions, but all of the non-vaccinated goats developed pyrexia and 5/9 developed neurological clinical signs, primarily tremors in the neck and ataxia. All non-vaccinated animals developed histological lesions restricted to the central nervous system and consistent with a lymphocytic meningomyeloencephalitis. Vaccinated goats had significantly (P <0.003) greater concentrations of serum IgG and lower levels of IgM (P <0.0001) compared with unvaccinated animals. SGEV RNA levels were below detectable limits in the vaccinated goats throughout the experiment, but increased rapidly and were significantly (P <0.0001) greater 2-10 days post challenge in the non-vaccinated group. In conclusion, vaccination of goats against LIV confers highly effective protection against SGEV; this is probably mediated by IgG and prevents an increase in viral RNA load in serum such that vaccinated animals would not be an effective reservoir of the virus.

Lambs are Susceptible to Experimental Challenge with Spanish Goat Encephalitis Virus.

Spanish goat encephalitis virus (SGEV) is a member of the genus Flavivirus, family Flaviviridae, and causes encephalomyelitis in goats. The aim of this study was to determine whether sheep are susceptible to experimental challenge with SGEV by two different routes. The results show that SGEV can infect sheep by both the subcutaneous and intravenous routes, resulting in neurological clinical disease with extensive and severe histological lesions in the central nervous system. Lambs challenged subcutaneously developed more severe lesions on the ipsilateral side of the brain, but the lesion morphology was similar irrespective of the route of challenge. The clinical presentation, pathogenesis, lesion morphology and distribution shows that SGEV is very similar to louping ill virus (LIV) and therefore any disease control plan must take into account any host species and SGEV vectors as potential reservoirs. Furthermore, discriminatory diagnostics need to be applied to any sheep or goat suspected of disease due to any flavivirus in areas where SGEV and LIV co-exist.

Two ovine mitochondrial DNAs harboring a fifth 75/76 bp repeat motif without altered gene expression in Northern Spain.

The Basque Country is home to the Latxa sheep breed, which is divided in several varieties such as Latxa Black Face (LBKF) and Latxa Blonde Face (LBLF). Mitochondrial DNA control region analysis of 174 male sheep (97 LBKF and 77 LBLF) was performed with the objective of characterizing the maternal lineages of these two varieties that are the basis to produce the cheese with Idiazabal quality label. The percentage of unique haplotypes was 77.32% in LBKF and 67.53% in LBLF. Most of the individuals were classified into B haplogroup (98.85%), while A haplogroup was much less frequent. Two Latxa individuals (one LBKF and one LBLF), both belonging to B haplogroup, displayed an additional 75/76 bp tandem repeat motif. Only 33 other sequences with this repeat motif were found among 11 061 sheep sequences included in the GenBank database. Gene expression was analyzed in peripheral blood leukocytes since the additional 75/76 bp repeat motif falls within ETAS1, a domain with a possible function in regulation of replication and transcription. The mRNA expression from four mitochondrial genes (COI, cyt b, ND1, and ND2) was analyzed in the two individuals of this study with a fifth repeat motif and in four without it. Although lower transcription was observed when the additional 75/76 bp repeat motif was present, no statistically significant differences were observed. Therefore, the variation in the number of the 75/76 repeat motif does not seem to modify the gene expression rate in mitochondrial genes.

Mycobacterial Infections in Rabbits: From the Wild to the Laboratory.

Tuberculous mycobacterial diseases such as leprosy and tuberculosis are ancient diseases that currently continue threatening human health in some countries. Non-tuberculous mycobacterial (NTM) infections cause a series of well-defined pathological entities, as well as some opportunistic diseases that have also increased worldwide, being more common among immunocompromised patients but rising also in immunocompetent individuals. Reports on natural infections by mycobacteria in rabbits are scarce and mainly involve NTM such as Mycobacterium avium subsp. avium in pigmy rabbits in the United States and Mycobacterium avium subsp. paratuberculosis in wild rabbits in Europe. Rabbits have been used as laboratory animals through the years, both to generate immunological reagents and as infection models. Mycobacterial infection models have been developed in this animal species showing different susceptibility patterns to mycobacteria in laboratory conditions. The latent tuberculosis model and the cavitary tuberculosis model have been widely used to elucidate pathogenic mechanisms and to evaluate chemotherapy and vaccination strategies. Rabbits have also been used as bovine paratuberculosis infection models. This review aimed to gather both wildlife and experimental infection data on mycobacteriosis in rabbits to assess their role in the spread of these infections as well as their potential use in the experimental study of mycobacterial pathogenesis and treatment.

Small ruminant lentivirus infections and diseases.

Small ruminant lentiviruses include viruses with diverse genotypes that frequently cross the species barrier between sheep and goats and that display a great genetic variability. These characteristics stress the need to consider the whole host range and to perform local surveillance of the viruses to opt for optimum diagnostic tests, in order to establish control programmes. In the absence of effective vaccines, a comprehensive knowledge of the epidemiology of these infections is of major importance to limit their spread. This article intends to cover these aspects and to summarise information related to characteristics of the viruses, pathogenesis of the infection and description of the various syndromes produced, as well as the diagnostic tools available, the mechanisms involved in transmission of the pathogens and, finally, the control strategies that have been designed until now, with remarks on the drawbacks and the advantages of each one. We conclude that there are many variables influencing the expected cost and benefits of control programs that must be evaluated, in order to put into practice measures that might lead to control of these infections.

SNPs in candidate genes MX dynamin-like GTPase and chemokine (C-C motif) receptor-5 are associated with ovine pulmonary adenocarcinoma progression in Latxa sheep.

Ovine pulmonary adenocarcinoma (OPA) is a contagious lung cancer in sheep caused by Jaagsiekte sheep retrovirus (JSRV). OPA is present in many sheep-rearing countries causing economic and welfare issues, as currently no efficient vaccines or treatments are available. Breed differences suggest a host genetic component may influence the pathogenesis of OPA, but so far few genes have been identified. In this work, a genetic association study was carried out in Latxa dairy sheep which were classified as cases/controls based on the presence/absence of OPA lung tumours. Candidate genes included cytokines and a receptor and innate immunity genes. After SNPs in the candidate genes were identified, the distribution of alleles in cases and controls was compared by means of logistic regression analyses at the allelic, genotypic and haplotypic levels. The association analysis showed that several candidate genes were significantly associated with resistance or susceptibility to OPA; two of the candidates, CCR5 and MX1, remained significantly associated with resistance and susceptibility respectively, even after Bonferroni correction.

SNPs in APOBEC3 cytosine deaminases and their association with Visna/Maedi disease progression.

The Apolipoprotein B mRNA-editing catalytic polypeptide-like 3 (APOBEC3) genes are able to inhibit the replication of a wide range of exogenous retroviruses, as well as endogenous retroviruses and retrotransposons. Three APOBEC3 genes, named APOBEC3Z1, APOBEC3Z2 and APOBEC3Z3, have been described in sheep. In this work the three genes have been screened in order to identify polymorphisms. No polymorphism was detected for the A3Z2 and A3Z3 genes but 16 SNPs and a 3-bp deletion were found in the A3Z1 gene. A thermoestability prediction analysis was applied to the detected amino acidic SNPs by three different programs. This analysis revealed a number of polymorphisms that could affect the protein stability. The SNPs of the 3'UTR were tested to detect alterations on the predicted microRNA target sites. Two new microRNA target sites were discovered for one of the alleles. Two SNPs were selected for association studies in relation with the retroviral disease Visna/Maedi in Latxa and Assaf sheep breeds. Although association analyses resulted unconclusive, probably due to the unsuitability of the SNP allele frequency distribution of the selected polymorphisms in the analyzed breeds, these genes remain good candidates for association studies.

Pathological findings in young and adult sheep following experimental infection with 2 different doses of Mycobacterium avium subspecies paratuberculosis.

Mycobacterium avium subsp paratuberculosis (Map) is assumed to infect young ruminants; however, little is known concerning the possibility of adult animals becoming infected. An experimental infection was conducted to establish the effect of age and doses of Map on susceptibility to paratuberculosis in sheep. Sixteen of twenty-four 1.5-month-old Churra lambs and 23 of 30 adult ewes (from 2-11 years old) were orally challenged with an ovine field strain of Map. Thirteen ewes and 8 lambs were infected with a high dose (HD) and 10 adult sheep and 8 lambs with a low dose (LD) of Map. The remaining animals were unchallenged controls. Animals were euthanized at 110 to 120 and 210 to 220 days postinfection. Histological, bacteriological, and nested polymerase chain reaction (PCR) studies were conducted in samples of intestine and related lymphoid tissue (Peyer patches, lymph nodes). Animals were classified according to their lesions. The number of granulomas was counted in 3 tissue sections from each sample. Only the HD groups showed lesions associated with paratuberculosis (92.3% of ewes and 100% of lambs). Adults had lesions characterized by few small demarcated focal granulomas restricted to the lymphoid tissue, whereas granulomas were more numerous and larger, appearing in the lamina propria unrelated to lymphoid tissue, in the lambs. Only HD-infected lambs were positive to culture, whereas nested PCR also detected positive HD ewes and some LD animals. These results suggest that adult sheep can become infected by Map, as seen by the development of lesions, but they are focal and restricted to the lymphoid tissue.

Pathological and aetiological studies in sheep exhibiting extrathoracic metastasis of ovine pulmonary adenocarcinoma (Jaagsiekte).

Seven sheep with a histopathological diagnosis of pulmonary adenocarcinoma with extrathoracic metastases were included in this retrospective study aiming to describe the pathological findings and to establish their relationship with Jaagsiekte sheep retrovirus (JSRV). In order of frequency, extrathoracic metastases were found in the liver, kidneys, skeletal muscle, digestive tract, spleen, skin and adrenal glands. Intrathoracic metastases involved the chest wall, regional lymph nodes, diaphragm and heart. Immunohistochemistry and polymerase chain reaction allowed detection of JSRV-related protein and nucleic acid, respectively, in the extrathoracic tumours of all cases. It is concluded that extrathoracic metastases constitute a pathological event of ovine pulmonary adenocarcinoma and confirm the malignant character of this virus-induced neoplasia.

Monitoring piroplasms infection in three cattle farms in Minorca (Balearic Islands, Spain) with previous history of clinical piroplamosis.

Bovine piroplasmosis are tick-borne protozoan diseases caused by parasites of the genera Theileria and Babesia. Three Friesian cattle farms (F1-F3) with previous history of clinical piroplamosis were selected in Minorca (Balearic Islands, Spain). Blood samples were collected from 8 to 11 animals every two months throughout a year and, a newly developed multiplex DNA bead-based suspension array based on the Luminex(®) xMAP technology was used to monitor for the presence of piroplasms. The assay incorporated probes for Babesia divergens, Babesia bovis, Babesia bigemina, Babesia major, Babesia occultans, Theileria annulata and Theileria buffeli, and a Catch-all Theileria and Babesia (TB) control probe. An internal amplification control that was detected with a Luminex probe was also included to monitor for inhibition. Infection was detected in 87.5% of the samples, 38.7% as single infections and 48.8% as mixed infections. T. annulata was widespread in Farm F1, with all animals positive over the whole study; albeit less frequently, T. annulata was also detected in Farms F2 and F3. T. buffeli was the overall most prevalent piroplasm, with a wide distribution in Farms F2 and F3 but only occasionally detected in F1. B. bigemina was the most frequent Babesia species, but was absent from Farm F1. B. bovis, previously reported in Minorca, was only sporadically detected in F2 and F3. A further 3 Babesia species not previously found in Minorca were also identified: B. major present in the 3 farms; B. divergens detected once in 2 animals in F2; and B. occultans found in 4 animals in F2 and in 1 Hyalomma marginatum female tick collected from a positive animal. Sequencing confirmed the identity of B. occultans thus extending the distribution of this species to Mediterranean Europe. This study confirmed the endemic situation for piroplasm infection in the region and detected the presence of a large number of chronic asymptomatic carriers. More importantly, 3 Babesia species not previously detected in the region were detected for the first time.

Microsatellites in immune-relevant regions and their associations with Maedi-Visna and ovine pulmonary adenocarcinoma viral diseases.

Maedi-Visna (MV) and ovine pulmonary adenocarcinoma (OPA) are two retroviral diseases occurring worldwide that affect adult sheep. Differences in incidence, which may be related to sheep-rearing and housing choices, as well as to genetics, and disease progression have been reported for both diseases. In this work four microsatellites located in immune-relevant regions, the major histocompatibility complex (MHC) region, interferon-γ and interleukin-12p35, were genotyped to determine their association with disease progression. The analysed sample included Latxa sheep with and without OPA and MV-characteristic lesions in their lungs. The microsatellites in the MHC were the most diverse, while the ones located in the cytokines were the less polymorphic. In the case of IFN-γ the results suggested the presence of null alleles. Significant results were detected for several microsatellite alleles in the association analysis carried out by logistic regression. All statistical analyses included a flock effect adjustment to avoid false positives due to genetic structuration. MHC Class I microsatellite alleles OMHC1*205 and OMHC1*193 were associated with disease progression for Maedi and OPA, respectively. Moreover, MHC Class II microsatellite allele DRB2*275 was associated with presence of lesions in Maedi. Furthermore, the MHC microsatellites were combined for a bioinformatic haplotype inference with the PHASE software. In total, 73 haplotypes were detected, 18 of them in more than 6 animals. After standard and weighted logistic regression analysis, two of them were significantly associated with susceptibility: OMHC1*205-DRB2*271 for Maedi and OMHC1*193-DRB2*271 for OPA, both with the Class I microsatellite alleles associated in the marker by marker study. Although more extensive analyses are needed to disentangle the relationship between host genetics and disease, as far as we know this is the first study demonstrating a significant association between sheep MHC Class I microsatellite alleles and susceptibility to Maedi-Visna and OPA viral diseases.

Immunization of adult dairy cattle with a new heat-killed vaccine is associated with longer productive life prior to cows being sent to slaughter with suspected paratuberculosis.

The objective of this study was to evaluate the efficacy of a new heat-killed Mycobacterium avium ssp. paratuberculosis (MAP) vaccine for control of premature culling in tuberculosis-free dairy cattle. Feces and gastrointestinal tissues were collected from 50 vaccinated cows and 38 nonvaccinated cows at slaughter and analyzed by bacteriological culture and histopathology. Vaccination was associated with a significant reduction of the frequency of vaccinated animals with MAP in feces and gut tissues compared with the nonvaccinated animals. In addition, the frequency of vaccinated animals with heavy bacterial load in gut tissues was 40% lower than the frequency of the nonvaccinated animals with the same MAP load. The peak age of paratuberculosis-associated culling was from 4.5 to 5 yr old (21%) in the vaccinated animals and from 3 to 4.5 yr old (60%) in the nonvaccinated animals. The vaccinated and nonvaccinated animals with suspected paratuberculosis were culled at an average age of 4.7 and 3.7 yr old, respectively. Therefore, a significant increase in the productive life of the vaccinated animals sent to slaughter with suspected paratuberculosis was observed. In addition, our analysis revealed a positive effect of the vaccine on the carcass weights of the animals with severe histopathological lesions at slaughter compared with the nonvaccinated animals. In summary, our findings suggest a therapeutic effect of the vaccine and a significant attenuation of pre-existing infection in cows naturally infected with paratuberculosis that were adults at the time of vaccination.

Presence of Bartonella species in wild carnivores of northern Spain.

The genus Bartonella was detected by PCR in 5.7% (12/212) of wild carnivores from Northern Spain. Based on hybridization and sequence analyses, Bartonella henselae was identified in a wildcat (Felis silvestris), Bartonella rochalimae in a red fox (Vulpes vulpes) and in a wolf (Canis lupus), and Bartonella sp. in badgers (Meles meles).

Genetic variation of toll-like receptor genes and infection by Mycobacterium avium ssp. paratuberculosis in Holstein-Friesian cattle.

Toll-like receptors (TLR) are membrane proteins that play a key role in innate immunity, by recognizing pathogens and subsequently activating appropriate responses. Mutations in TLR genes are associated with susceptibility to inflammatory and infectious diseases in humans. In cattle, 3 members of the TLR family, TLR1, TLR2, and TLR4, are associated with Mycobacterium avium ssp. paratuberculosis infection, although the extent of this association for the TLR1 and TLR4 receptors has not yet been determined. Moreover, the causal variant in the TLR2 gene has not yet been unequivocally established. In this study, 24 single nucleotide polymorphisms (SNP) in the bovine TLR1, TLR2, and TLR4 genes were selected from the literature, databases, and in silico searches, for a population-based genetic association study of a Spanish Holstein-Friesian sample. Whereas previous results regarding the TLR1 gene were not corroborated, a risk haplotype was detected in TLR2; however, its low frequency indicates that this detected association should be interpreted with caution. In the case of the TLR4 gene, 3 tightly linked SNP were found to be associated with susceptibility to M. avium ssp. paratuberculosis infection. Moreover, one of these SNP, the SNP c.-226G>C, which is localized in the 5'UTR region of the TLR4 gene, has been reported to be able to alter TLR4 expression, raising the possibility that this mutation may contribute to the response of the individual to infection.

Differences in questing tick species distribution between Atlantic and continental climate regions in Spain.

Climate and vegetation in Spain vary from north to south, affecting tick distribution and consequently the presence of tick-borne diseases. The aim of this study was to investigate throughout a 2-yr study the distribution of the different exophilic questing tick species present in 18 areas: eight located in central and 10 in northern Spain. The same methodology was used in both areas, sampling vegetation on a monthly basis by blanket dragging for 20- to 30-min intervals. A total of 12 species belonging to the genera Ixodes, Haemaphysalis, Rhipicephalus, Dermacentor, and Hyalomma was identified. Differences in species distribution and prevalence were dramatically different. The most frequent and abundant species in northern Spain were Ixodes ricinus (67% of adult ticks) and Haemaphysalis punctata (8%), whereas Hyalomma lusitanicum (86%) and Dermacentor marginatus (12%) were the most abundant in central Spain. There were important differences in the monthly seasonal patterns for the different tick species. These results highlight important differences in tick distribution in neighboring areas and underline the need for ongoing surveillance programs to monitor tick population dynamics and the prevalence of tick-borne pathogens.

Measuring antibody levels in bulk-tank milk as an epidemiological tool to search for the status of Coxiella burnetii in dairy sheep.

Pooling samples may provide a valuable alternative to individual testing for pathogen surveillance purposes. We studied the reliability of measuring the level of antibodies against Coxiella burnetii in bulk-tank milk (BTM) to estimate the seroprevalence of C. burnetii in dairy sheep in 34 flocks. We then estimated the seroprevalence of C. burnetii in 154 dairy sheep flocks according to the level of antibodies in BTM. We tested for the accuracy of our estimation at the population level by comparing predicted mean C. burnetii flock seroprevalence with that obtained in another survey performed on the same population. Our findings showed that testing BTM by ELISA is a cost-effective and relatively good index of the seroprevalence of C. burnetii in dairy sheep and may be a useful tool for epidemiological surveillance at the population level.