Serum haptoglobin response in red deer naturally infected with tuberculosis.

The analysis of haptoglobin (Hp) serum concentration is a very sensitive, but non-specific, indicator of inflammation or infection. Methods to accurately diagnose infection in vivo in wildlife are usually constrained by low sensitivity due to the effects of stress on individual immune response and the challenging logistics of performing tests in the wild. Firstly, we sought to determine serum Hp concentration in red deer (Cervus elaphus) naturally infected with bovine tuberculosis (TB). Secondly, we assessed the complementary diagnostic value of serum Hp levels in conjunction with the cervical comparative skin test (CCT) performed in a subsample (n = 33). Serum Hp concentrations were significantly higher in TB-infected individuals (based on the presence of macroscopic lesions confirmed by culture) compared to those uninfected. In addition, serum Hp significantly changed with the type of animal handling, with captured and handled animals showing higher levels of Hp than hunted animals. Four out of 6 TB positive individuals that tested negative to the CCT (false negatives) showed Hp levels higher than the 95th percentile of healthy animals. These findings indicate that an acute phase response develops in animals with TB. In this paper, we demonstrate for the first time that an acute phase protein can provide a complementary assessment for specific diagnosis tests in wild species.

Biotic and abiotic factors shape the microbiota of wild-caught populations of the arbovirus vector Culicoides imicola.

Biting midges of the genus Culicoides are known vectors of arboviruses affecting human and animal health. However, little is known about Culicoides imicola microbiota and its influence on this insect's biology. In this study, the impact of biotic and abiotic factors on C. imicola microbiota was characterized using shotgun-metagenomic sequencing of whole-body DNA samples. Wild-caught C. imicola adult nulliparous females were sampled in two locations from Sicily, Italy. The climatic variables of temperature and soil moisture from both localities were recorded together with potential host bloodmeal sources. Shared core microbiome among C. imicola populations included Pseudomonas, Escherichia, Halomonas, Candidatus Zinderia, Propionibacterium, and Schizosaccharomyces. Specific and unique taxa were also found in C. imicola from each location, highlighting similarities and differences in microbiome composition between the two populations. DNA and protein identification showed differences in host preferences between the two populations, with Homo sapiens and Canis lupus familiaris L. being the preferred bloodmeal source in both locations. A principal component analysis showed that the combined effect of host preferences (H. sapiens) and local soil moisture factors shape the microbiome composition of wild-caught populations of C. imicola. These results contribute to characterizing the role of the microbiome in insect adaptation and its utility in predicting geographic expansion of Culicoides species with potential implications for the control of vector-borne diseases.

Sex-related differences in body condition and serum biochemical parameters in red deer (Cervus elaphus) naturally infected with Mycobacterium bovis.

Although Mycobacterium bovis infection is commonly reported in red deer (Cervus elaphus), potential differences in the effects of infection on male and female animals in terms of body condition and clinical biochemistry have not been reported. Between November 2000 and January 2006, serum and biometrical data were collected post-mortem from 88 red deer. M. bovis-infected deer, particularly males, were typically older, heavier and in poorer body condition than uninfected animals. Serum triglyceride, cholesterol (both particularly in males) and total protein concentrations were lower, whereas serum creatinine (more evident in females), and immunoglobulin G and M concentrations were higher in the infected deer. These sex-related differences in the response to M. bovis infection in red deer should be considered when undertaking epidemiological assessments and designing disease control strategies as they may reflect differing roles of male and female animals as potential reservoirs or disseminators of disease.

Prevalence of tick-borne pathogens in adult Dermacentor spp. ticks from nine collection sites in France.

The importance of Dermacentor spp. in the transmission of tick-borne pathogens is not well recognized in Europe. To investigate the role of Dermacentor spp. in the transmission of tick-borne pathogens, questing ticks were collected in 9 sites from southern to northwestern France (Camargue Delta to Eastern Brittany) where Dermacentor spp. exist and tick-borne diseases had occurred previously. Three tick species were collected during the spring and autumn of 2009. Collected ticks (both males and females) included D. marginatus (n=377), D. reticulatus (n=74), and I. ricinus (n=45). All ticks were analyzed by PCR or reverse line blot for the presence of pathogens' DNA. Pathogens analyzed were based on veterinarian reports and included Anaplasma phagocytophilum, Coxiella burnetii, Anaplasma marginale, Borrelia burgdorferi, Bartonella spp., Babesia spp., Theileria spp., and Francisella sp. Francisella tularensis was not detected in any of the analyzed ticks. In D. marginatus, infection prevalence for A. phagocytophilum (3%) was similar to that found in I. ricinus in Europe. Other pathogens present in D. marginatus included A. marginale (0.5%), Bartonella spp. (9%), C. burnetii (12%), F. philomiragia (1.3%), and Theileria annulata/Babesia bovis (0.3%), which were detected for the first time in France. Pathogens detected in D. reticulatus included A. marginale (1%), Bartonella spp. (12%), C. burnetii (16%), Borrelia spp. (1.5%), and F. philomiragia (19%). Pathogens detected in I. ricinus included A. phagocytophilum (41%), Bartonella spp. (9%), C. burnetii (18%), A. marginale (1%), Borrelia spp. (4.5%), and Babesia sp. (7%). This study represents the first epidemiological approach to characterize tick-borne pathogens infecting Dermacentor spp. in France and that may be transmitted by ticks from this genus. Further experiments using experimental infections and transmission may be now conducted to analyze vector competency of Dermacentor spp. for these pathogens and to validate such hypothesis.

Fatal bovine anaplasmosis in a herd with new genotypes of Anaplasma marginale, Anaplasma ovis and concurrent haemoplasmosis.

Haematological and molecular analysis of blood samples was carried out during an outbreak of bovine anaplasmosis in Hungary. Acute disease was observed in five animals, two of which died. Anaplasma-carrier state was diagnosed in 69 (92%) of cattle. Further evaluation of 24 blood samples revealed concurrent infections with Mycoplasma wenyonii and 'CandidatusM. haemobos' in 22 and 21 animals, respectively. In addition, two cows were identified with rickettsaemia. Regarding molecular investigation of potential hard tick vectors, Haemaphysalis inermis and Dermacentor marginatus males collected from the animals were PCR-negative. However, in one pool (out of 18) of Ixodesricinus males, and in six pools (out of 18) of D. reticulatus males the msp4 gene of Anaplasma marginale was detected. In the same I. ricinus pool Anaplasma ovis was also identified. All ticks were negative for haemoplasmas. Anaplasma sequences yielded 97-99% homology to sequences deposited in the Genbank. This is the first report of fatal bovine anaplasmosis associated with divergent A. marginale genotypes and concurrent 'CandidatusM. haemobos' infection, as well as of an A. ovis strain in ticks collected from cattle.

Diagnosis of tuberculosis in camelids: old problems, current solutions and future challenges.

In spite of great efforts for its control and eradication, tuberculosis remains one of the most important zoonosis worldwide. Its causative agents, the members of the Mycobacterium tuberculosis complex, have a wide host range that complicates the epidemiology of this disease. Among susceptible species to these pathogens, camelids from the New World (llama, alpaca and vicuña) and Old World (Bactrian camel and dromedary) are acquiring an increasing importance in several European countries because of its growing number and could act as reservoirs of the disease for livestock and humans in their natural habitat. In addition, tuberculosis caused by a number of M. tuberculosis complex members is a life-threatening disease in these animal species. Although tuberculosis has been known to affect camelids for a long time, ante-mortem diagnosis is still challenging because of the lack of standardized diagnostic techniques and the limited sensitivity and specificity of the most widely applied tests. However, in recent years, several techniques that can at least partially overcome these limitations have been developed. This paper reviews the results and advances achieved in tuberculosis diagnosis in camelids in the last decade as well as the progresses on ongoing investigations, with special attention to the remaining challenges that still have to be faced to assure the availability of reliable tools for the detection of tuberculosis-infected animals and herds.

Unexpected high responses to tuberculin skin-test in farmed red deer: implications for tuberculosis control.

Tuberculosis (TB) in deer is a serious zoonotic disease of worldwide distribution. Detection of infected animals is usually performed using single or comparative skin-testing (SST/CST), although false responses due to sensitization to other mycobacteria may occur, hampering diagnostic specificity. We describe the evolution of the responses to the SST, CST and to an in-house serological assay in a red deer farm subjected to regular TB testing in southern Spain in an attempt to understand the dynamics of possible non-specific reactions occurring under field conditions. We performed 2288 skin-tests and ELISAs in nine sampling periods between May 2009 and January 2011. In May 2010, a strong increase in skin fold thickness in response to avian purified protein derivative (PPD) (mean=4.0mm, 95% CI=3.5-4.5) and bovine PPD (mean=1.8mm, 95% CI=1.6-2.0) was observed in yearling deer hinds (n=150), compared to values recorded for the same individuals in November 2009 (avian PPD: mean=0.7 mm, 95% CI=0.6-0.8 and bovine PPD: mean=0.7 mm, 95% CI=0.6-0.7) and in January 2011 (avian PPD: mean=2.2mm, 95% CI=1.9-2.4 and bovine PPD: mean=1.1mm, 95% CI=1.0-1.2). Using SST, 54 animals (36%) of the yearlings tested in May 2010 would have been classified as positive reactors, while none of them was positive in the CST. The five animals with highest skin fold increases to mycobacterial antigens were culled and subjected to post-mortem analysis, which confirmed the absence of Mycobacterium tuberculosis complex (MTBC) infection but demonstrated the presence of environmental mycobacteria and closely related bacteria in four out of the five analyzed animals. Our results demonstrated how non-specific responses to mycobacterial antigens can adversely affect the specificity of TB diagnosis based on the SST. Thus, once TB infection has been ruled out using confirmatory techniques, application of comparative diagnostic tests is highly advisable to maximize test specificity and avoid the slaughter of false positive reactors.

No evidence that wild red deer (Cervus elaphus) on the Iberian Peninsula are a reservoir of Mycobacterium avium subspecies paratuberculosis infection.

The potential role of red deer (Cervus elaphus) as a reservoir of Mycobacterium avium subspecies paratuberculosis (MAP) infection is largely unknown. A total of 332 wild red deer were investigated using post-mortem examination, bacteriology and serology. Only three animals (1.12%) were found to have lesions on histopathological examination and no MAP bacteria were recovered on culture. The results suggest it is unlikely that wild red deer make a significant contribution to the maintenance of MAP infection in the region. The cross-reactivity of the ELISAs used indicates this diagnostic modality is ineffective in the detection of MAP infection in this species. The implications of these results for the control of this important pathogen in both livestock and wildlife are discussed.

The testing season affects red deer skinfold increase in response to phytohaemagglutinin.

Red deer (Cervus elaphus) have a pronounced seasonality in their physiology. The aim of this study was to assess the effect of the season on red deer responsiveness to skin testing with the phytohaemagglutinin (PHA) mitogen. Study subjects included 270 farmed adult red deer (19 stags and 251 hinds). The skin testing was carried out between January 2009 and August 2010. The animals were injected intradermally with a 0.1 ml volume containing 250 µg of PHA diluted in phosphate buffered saline. The skinfold thickness was measured immediately prior to injection and 72 h after administration, always by the same person and with three repeats per measurement. Single effects of sex and time on skin test responsiveness were significant (p < 0.001) as well as their interaction (p < 0.001). In winter (January), and considering the average of two years, the skinfold increase in response to the intradermal injection of 250 µg PHA was 2.1 times larger in stags and 1.4 times in hinds than in summer (August). While stags had 1.3 times larger responses than hinds in winter, the inverse occurred in summer, with 1.1 times larger responses in hinds. We also evidenced a limited inter-annual variation of skinfold increase in response to PHA in red deer. These findings have important consequences regarding the interpretation of skin test results in the ante-mortem diagnosis of tuberculosis and paratuberculosis, at least in deer.

Eurasian wild boar response to skin-testing with mycobacterial and non-mycobacterial antigens.

Eurasian wild boar (Sus scrofa) are able to maintain bovine tuberculosis (bTB) in the wild and are most probably able to transmit the disease to other species, thus acting as a true wildlife reservoir. Translocation of wild boar is a common practice in European countries. Therefore, identifying effective tools for bTB diagnosis in living wild boar is crucial for the implementation of control measures. We describe for the first time the sex and origin related differences in the skin-test response to mycobacterial antigens (bPPD and aPPD) and to a non-mycobacterial antigen (PHA, a plant derived mitogen) in wild and farmed wild boar, and used a small sample of known M. bovis infected wild boar to establish whether skin-testing is an option for bTB diagnosis in living wild boar. The highest skinfold increase response was detected at the PHA injection site, evidencing that the PHA test could be useful in monitoring cell mediated immunity (CMI) in wild boar populations. A clear age-increasing trend of the PHA response indicated that age should be taken into account when measuring CMI in wild boar. Origin related differences in the response against mycobacterial antigens could reflect differential exposure to mycobacterial antigens. Skin testing in BCG immunized wild boar showed low sensitivity (43-57%), while the sensitivity was good in the culture positive controls (75-100%), depending on the reading criterion. Specificity improved when the criterion was a response to bPPD larger than 2 mm and bPPD response larger than aPPD response (77%). Although a limited sample, our results indicated the potential of skin test as a bTB diagnostic tool in Eurasian wild boar. However, handling of wild boar is dangerous, specificity is low, and more effort is needed in order to define the sensitivity of this technique.

Factors affecting red deer skin test responsiveness to bovine and avian tuberculin and to phytohaemagglutinin.

We studied the effect of management on the responsiveness of red deer (Cervus elaphus) to skin testing with mycobacterial and non-mycobacterial antigens. We hypothesized that individuals from populations of the same species under different management conditions would have a different immune responsiveness. Deer sampled in this study included 1041 adult animals from 6 Spanish farms and 111 adult wild deer. We injected four sites of the neck with 0.1 ml bovine purified protein derivative (PPD), 0.1 ml avian PPD, 0.1 ml negative control PBS and 0.1 ml of Phytohaemagglutinin (PHA, containing 250 microg) as positive control, and measured the skin fold increase at time 72 h. Bovine PPD reactors were identified in 5 of 6 farms and among wild deer. Apparent prevalence among wild deer (18.9%) was not significantly higher than among farmed deer (14.5%). Avian PPD reactors were found among all 7 study populations, but apparent prevalence was lower among wild deer (<1%) than among farmed deer (12.6%; p<0.001). Deer management (farmed versus wild) was identified as a key factor affecting deer skin fold thickness increase in response both to mycobacterial (bPPD and aPPD) and non-mycobacterial antigens (PHA). The differences occurred in the same sense, regardless of some interactions; farmed deer showing higher values. The PHA skin fold increase was not affected by the PPD skin test results. We propose that using PHA as a positive control may help in the interpretation of between-population differences in tuberculin responses.

The effects of sex and age on phytohaemagglutinin skin-testing of deer.

AIM: To determine if there are sex- or age-related differences in the increase in skinfold thickness in response to the mitogen phytohaemagglutinin (PHA) in red deer. METHODS: One dose of 250 mug PHA was injected intradermally in the right side of the neck, and phosphate buffered saline (PBS) was injected at a second site as a control, in 110 (51 males and 59 females) captive Iberian red deer (Cervus elaphus hispanicus), ranging in age from 21 months to > or =5 years. Skinfold thicknesses were measured immediately before and 72 h following injection. RESULTS: There was a significant effect of gender on the average increase in skinfold thickness; males had greater increases (8.8 (SEM 0.57) mm) than females (4.23 (SEM 0.39) mm) after correcting for other confounding variables. No age-related differences were evident, but differences between sexes were more marked with increasing age. CONCLUSIONS AND CLINICAL RELEVANCE: Effects of gender, probably due to differences in energetic and reproductive constraints in red deer, should be taken into account when interpreting skinfold-test data, both in ecology and in the control of tuberculosis (Tb). Males tend to have a thicker skin than females, so skinfold increase relative to the thickness of the skin, rather than skinfold increase per se, should be used as a more appropriate measure of skinfold increase. This may also have clinical relevance in the interpretation of tuberculin skin testing.

Optimal dose and timing in phytohaemagglutinin skin-testing of deer.

AIM: To establish the optimal dose of the mitogen phytohaemagglutinin (PHA) and the optimal time for measuring increased skin-fold thickness in red deer following intradermal injection, as an indicator of cell-mediated immune response. METHODS: Three doses (10, 50 and 250 microg) of PHA were injected intradermally in the right side of the neck, and phosphate buffered saline (PBS) was injected at a fourth site as a control, in 20 captive Iberian red deer (Cervus elaphus hispanicus) hinds. Skin-fold thicknesses were measured at 0, 12, 24, 36, 48, 60, 72, 84 and 96 h following injection. RESULTS: The highest dose of PHA tested (250 microg) resulted in a clear and long-lasting cellular response; increases in skin-fold thickness between 48 and 84 h post-injection varied minimally and response correlated positively with liveweight. No correlations with liveweight and no clear increases in skin-fold thickness occurred at the lower doses of PHA or the PBS. CONCLUSIONS AND CLINICAL RELEVANCE: This technique could be applied with minimal training and without specialised equipment in deer, for immunological and ecological research.

Serosurvey of Aujeszky's disease virus infection in European wild boar in Spain.

Serum samples from 693 hunted wild boar (Sus scrofa) were analysed by means of a blocking ELISA technique, and the mean (se) prevalence of antibodies to Aujeszky's disease virus was 44 (4) per cent. All the seropositive wild boar were from south central Spain, except for one from central Spain, close to the main positive area. In this area, where large game species are increasingly managed for hunting, the seroprevalence was affected by the type of management. More intensively managed populations had a higher prevalence than wild boar living in natural situations, and the seroprevalence increased with the age of the animals; the seroprevalence was higher in females in all age groups. The seroprevalence in males more than one year old peaked after the breeding season, whereas females of the same age had a higher and constant seroprevalence throughout the year.