Frequency of antibodies against Besnoitia besnoiti in Brazilian cattle

Besnoitia besnoiti is a cyst-forming parasite that has been associated with economic losses in Africa and Europe. Besnoitiosis is considered as a re-emergent disease in the European continent. It is unknown whether cattle are exposed to B. besnoiti in the Americas, thus the aim of this study was to serologically investigate antibodies against B. besnoiti in a total of 2014 cattle serum samples from two states from Brazil. All samples were evaluated by IFAT and part of the positive sera was tested by Western blot (WB) using tachyzoites extracts under non-reducing condition. A total of 3.48% (70/2014) of the tested sera reacted positively by IFAT with titers of 200 (85.7%), 400 (10%) and 800 (4.3%). When 47 positive samples were assessed by WB a range of antigens from 7 to 206 kDa was recognized by the IFAT-positive sera. The results are suggestive of exposure of Brazilian cattle to B. besnoiti due to the titers (≥200) observed for some sera using IFAT. However, the antigens recognized by the IFAT-positive animals did not completely match with the WB patterns previously described by other working groups. It is possible that Brazilian cattle are exposed to B. besnoiti strains with different antigenic composition of those described in the European and African continent. Further studies are needed to confirm the presence of B. besnoiti or other Besnoitia species in Brazilian cattle.     

Blood-feeding patterns of horse flies in the French Pyrenees

Horse flies can mechanically transmit Besnoitia besnoiti, the agent of bovine besnoitiosis. Although previously limited to enzootic areas, especially the French Pyrenees Mountains, bovine besnoitiosis is now considered a re-emerging disease in western Europe. To improve understanding of the role of horse flies as mechanical vectors, this study investigated their blood-feeding ecology in the eastern French Pyrenees, in two high-altitude summer pastures whose main domestic ungulates were cattle, and in a wildlife park with native fauna. Species-specific PCR assays were conducted to identify the sources of blood meals: wild boar, horse, cattle (or bison), sheep (or mouflon), goat, red deer, roe deer and izard (or Pyrenean chamois). In La Mouline pasture, tabanids (N = 20) fed on red deer (70%) and cattle (30%). In Mantet pasture, tabanids (N = 24) fed on cattle (52%), red deer (20%), wild boar (16%), horse (8%) and sheep (4%). In the wildlife park, Tabanus bromius (N = 32), the most abundant species collected, fed on red deer (85%), bison (9%) and wild boar (6%). Despite relatively high densities in both the pastures and in the wildlife park, small wild ungulates (izard, mouflon and roe deer) were not detected as a source of blood meals. Only two mixed blood meals were identified in two specimens of T. bromius: cattle/horse for the specimen collected in the pastures, and bison/wild boar for the specimen collected in the wildlife park. Our findings showed that tabanids display a level of opportunistic feeding behaviour, in addition to a preference for red deer, the latter being particularly true for Philipomyia aprica, the most abundant species collected in the pastures.     

Evaluation of serological cross-reactivity between canine visceral leishmaniasis and natural infection by Trypanosoma caninum

In order to evaluate if the presence of Trypanosoma caninum can lead to a confuse diagnosis of canine visceral leishmaniasis (CVL), we investigated the serological status of dogs infected by T. caninum and assessed the serological cross-reactivity with CVL. A set of 117 serum samples from dogs infected by T. caninum, Leishmania chagasi and not infected dogs (n = 39 in each group) was tested using commercial kits – indirect immunofluorescence (IFI-LVC), ELISA (EIE-LVC) and immunochromatographic test (DPP) – and in house tests with T. caninum (IIF-Tc and ELISA-Tc) and L. chagasi antigens (IIF-Lc and ELISA-Lc). IIF-Tc and ELISA-Tc presented sensitivity of 64.1% and 94.9% and specificity of 23.1% and 35.9%, respectively. The sensitivity of the IFI-LVC, EIE-LVC and DPP tests was 100% and the specificity was 70.5%, 68% and 97.5% respectively. The concordance between the tests was considered as satisfactory. The specificities of IFI-LVC, EIE-LVC and DPP were higher when the group Tc was excluded, with significant values for IFI-LVC (χ² = 4.36, P-value = 0.036), thus suggesting that the infection by T. caninum can confuse the diagnosis of CVL.     

Preliminary findings from an experimental study of caprine besnoitiosis in Kenya

Inoculation of cystozoites obtained from natural, chronic cases of caprine besnoitiosis produced clinical disease in goats but not in rabbits, mice, guinea pigs, hamsters, rats or cattle. Histological examination of tissue sections from the experimental animals showedBesnoitia cysts only in goats. This, together with field observations that cattle reared together with goats having besnoitiosis do not contract the disease, suggests that theBesnoitia species that infects goats in Kenya is host-specific and is notBesnoitia besnoiti. We suggest that the nameBesnoitia caprae be adopted for the caprine pathogen.     

Serological survey of Neospora caninum and Toxoplasma gondii in cattle (Bos indicus) and water buffaloes (Bubalus bubalis) in ten provinces of Brazil

The objective of this study was to determine the prevalence of antibodies to Neospora caninum and Toxoplasma gondii among 500 cattle (Bos indicus) and 500 buffaloes (Bubalus bubalis) using the indirect fluorescent antibody test (IFAT) technique. Blood samples from were collected from water buffalo and cattle in 10 municipalities in the northern region of Brazil. The frequency of cattle and water buffaloes seropositive for Neospora caninum in Pará state, Brazil, was 55% and 44%, respectively, and the frequency of cattle and water buffaloes seropositive for Toxoplasma gondii was 52% and 39%, respectively. Seropositivity for both N. caninum and T. gondii was detected in 10.6% of the cattle samples and 14.8% of the buffalo samples. The frequency of cattle positive for N. caninum and T. gondii was significantly (p < 0.05) higher than that of buffalo in two and three provinces, respectively. Buffaloes had a lower seroprevalence for N. caninum or T. gondii in all of the provinces studied. These results suggest that both species, when exposed to the same risks for N. caninum and T. gondii infection, have a high serological prevalence. Cattle showed a higher probability of being seropositive when exposed to the same risks for N. caninum and T. gondii. Our study, which included an extensive number of blood samples, provides important epidemiological information pertinent to buffalo production in tropical countries that can be used as a basis for disease-management practices in Latin America.     

Evidence for bovine besnoitiosis in Egypt—first serosurvey of Besnoitia besnoiti in cattle and water buffalo (Bubalus bubalis) in Egypt

The present study evaluated the presence of specific antibodies against Besnoitia besnoiti in cattle and water buffalo (Bubalus bubalis) in Egypt. Sera from cattle (n?=?216) and water buffaloes (n?=?133) collected from five different provinces of Egypt (Behera, Alexandria, Assuit, Gharbia, and Matrouh) were analyzed. Testing for B. besnoiti antibodies by PrioCHECK® Besnoitia Ab 2.0 ELISA initially identified 13.75 % (48 out of 349) of individual sera as positive at the manufacturer’s suggested cutoff threshold, 15 percent positivity (PP). Statistically significant associations between B. besnoiti prevalence, species, sex, age, and geographical distribution were observed. Seropositive animals were distributed in all of the provinces from which animals were sampled except Gharbia province. Assuit province showed the highest percentage of infection (30.76 %) followed by Matrouh, Alexandria, and Behera provinces (25, 16.29, and 9.6 %, respectively). The highest infection rate of B. besnoiti was significantly higher in cattle (17.13 %) than in water buffaloes (9.02 %). Positive cases were observed in all age categories. While the highest infection rate (17.13 %) was recorded in the age group 5–10 years followed by the age group 1–5 years (15.38 %), and only one positive case (1.58 %) was recorded in the age group less than 1 year. The highest infection rate of B. besnoiti infection was recorded in the female animals (14.95 %) followed by the male animals (8.33). This is the first report on the detection of B. besnoiti in cattle and water buffaloes in Egypt.     

Optimization of in-house ELISA based on recombinant major sperm protein (rMSP) of Dictyocaulus viviparus for the detection of lungworm infection in cattle

The aim of this study was to optimize an in-house ELISA based on a recombinant version of the major sperm protein (MSP) of Dictyocaulus viviparus for routine diagnosis of lungworm infection in cattle. A recombinant MSP (rMSP) was cloned into pGEX-6P-1 vector and expressed as a glutathione-S-transferase (GST) fusion protein in Escherichia coli BL21 (DE3) chemically competent cells. The product was then employed as capture antigen in an ELISA, and validated against 304 samples of known status (216 negative and 88 positive) in which the antibody levels in sera had also been measured earlier with a commercial ELISA kit (Ceditest® lungworm ELISA). The receiver operating characteristic (ROC) curve analysis of the ELISA results estimated the optimized diagnostic sensitivity and specificity as 97.7% (95% confidence interval [CI]: 91.9–99.7%) and 98.1% (CI: 95.3–99.5%), respectively. The results from the in-house rMSP-based ELISA were compared with results obtained on both fecal examination and the Ceditest® lungworm ELISA. Rising antibody levels in sera of experimentally infected calves were observed between 21 and 28 days post infection, when patency was also confirmed by the presence of larvae in feces. Notably, using the in-house rMSP-based ELISA infection was confirmed in calves shedding larvae approximately 3–4 weeks post inoculation, while using the Ceditest® lungworm ELISA those animals remained negative. Additionally, 251 sera samples from calves naturally exposed to the parasites on pasture were used to evaluate the test. In in-house rMSP-based ELISA no cross-reactions were observed with sera from calves infected with the gastrointestinal nematodes (Ostertagia ostertagi and Cooperia oncophora), even though the presence of eggs in the feces was confirmed. Overall, the in-house rMSP-based ELISA optimized in this study showed excellent diagnostic performance for detection of lungworm infection in cattle.     

Risk factors associated with Neospora caninum seropositivity in randomly sampled Canadian dairy cows and herds

Our objective was to determine cow- and herd-level risk factors associated with seropositivity for Neospora caninum in a large number of randomly selected Canadian dairy herds, controlling for important confounding variables and co-infections with bovine leukemia virus (BLV), bovine viral diarrhea virus (BVDV) and Mycobacterium avium subspecies paratuberculosis (MAP). Serum samples were obtained from 30 randomly selected cows, where available, in 240 herds using monthly milk testing, within 6 of 10 provinces, and these samples were tested for antibodies against BLV, MAP and N. caninum using commercially available ELISA test kits. Five unvaccinated cattle >6 months old from each herd were tested for antibodies to BVDV using virus neutralization. Most herd-level predictors were obtained through personal interviews with questionnaires administrated to each farm manager. A mixed logistic-regression model was built using N. caninum serostatus at the cow-level as the outcome variable, with herd as a random effect and province as a fixed effect. A BLV seropositive cow was 1.50 times more likely to be seropositive for N. caninum than a BLV-seronegative cow, and this was the only cow-level variable to remain in the final model. Regarding herd-level variables, with “no on-farm dogs” as the baseline, “presence of dogs but not known to eat placentas and/or fetuses” increased the odds of seropositivity for N. caninum by a factor of 1.66. For “presence of dogs known to eat placentas and/or fetuses”, the odds ratio (OR) was 2.75, demonstrating a dose–response relationship. “Using embryo transfer” (OR = 0.69), “asking for a BVDV-negative test before introducing an animal” (OR = 0.30), “using monensin in dry cows” (OR = 0.71), and “heifers having nose-to-nose contact with calves” (OR = 0.73) were all dichotomous variables negatively associated with seropositivity for N. caninum. “Number of milk cows on the farm” (OR = 0.99), and “area (acres) used for forage production” (OR = 0.99) were continuous variables negatively associated with N. caninum seropositivity.     

Serodiagnosis of bovine besnoitiosis by ELISA and immunofluorescence tests

Sera from non-infected cattle and cattle infected with Anaplasma, Babesia, Theileria and Sarcocystis were tested for antibodies to Besnoitia in ELISA and immunofluorescence tests (IFT) with Besnoitia besnoiti of blue wildebeest origin as antigen. Only 2 out of 86 sera gave false positive reactions in ELISA and none in the IFT, indicating a high specificity for the tests. Three-hundred-and-three bovine sera from 3 farms in an area endemic for besnoitiosis were similarly tested and the results were correlated with clinical findings based on visual inspection for typical symptoms and the presence of cysts in the scleral conjunctiva. Most of the positive tests were observed in cattle older than 1 year. Of the cases with scleral cysts, 68,7% were positive in the ELISA and 81,74% in the IFT. However, 45,74% (ELISA) and 49,47% (IFT) of the clinically negative cattle were clinically positive, indicating a high incidence of clinically inapparent infection. These results indicate a relatively low sensitivity for these serological tests. An unexpected finding was that the ELISA remained negative for at least 60 days after experimental infection of the cattle, the maximum period for which tests were done, whereas the IFT became positive. No antibodies against B. besnoiti could be found in human sera. Besnoitia jellisoni antigen gave positive results with B. besnoiti antibodies in ELISA, but not in the IFT.     

Neospora caninum DNA detection by TaqMan real-time PCR assay in experimentally infected pregnant heifers

Neosporosis has been considered the main cause of abortion between the first and the second trimester of pregnancy in cattle. Therefore, the objective of this study was to identify the presence of Neospora caninum DNA obtained from experimental models based on the evaluation of different areas of the fetal nervous system and organs from heifers previously inoculated with NC-1 after or before insemination. This study was performed with Hereford × Nelore (n = 29) heifers and all animals were considered free of diseases at the beginning of the experiment. All animals were bred by fixed-time artificial insemination (TAI) and allocated as follows: (a) seronegative heifers subjected to TAI (TAI, n = 9), (b) heifers infected with N. caninun 60 days prior to TAI (NC-1 + TAI, n = 9), and (c) heifers submitted to TAI and infected with N. caninum 60 days later (TAI + NC-1, n = 11). The pregnancy was confirmed by transrectal ultrasonography 35 days after TAI and evaluated every 30 days until the end of gestation. Fetuses were collected surgically at 170 days of gestation, and immediately necropsied to remove tissues aseptically. Samples of the central nervous system (CNS), heart, kidney, lung, liver, skeletal muscle and caruncle were collected for DNA extraction. Days of gestation at abortion and interval from abortion to first insemination were examined by Student's t-test. At 35 days of gestation the pregnancy rates in the group NC-1 + TAI (4/9, 44.4%) was lower than in the control group (8/9, 88.8%, P < 0.05). At 60 days, the pregnancy rates in the NC-1 + TAI group (0/4, 0%) was lower compared to TAI + NC-1 (5/7, 71.4%) and control (6/8, 75.0%) groups (P < 0.05). Animals from the group NC-1 + TAI were re-inseminated 60 days after the first TAI. After pregnancy losses throughout the study, 5 animals (TAI), 3 animals (NC-1 + TAI) and 5 animals (TAI + NC-1) maintained pregnancy until 170 days of gestation. TaqMan RT-PCR demonstrated the presence of N. caninum DNA in the medulla and right posterior cortex in 3 out of 5 fetuses from the TAI + NC-1 group. We concluded that heifers infected after TAI had a higher incidence of the parasite at the fetus CNS. Identification of N. caninum by TaqMan RT-PCR would assist in the investigation of infection and in the evaluation of vaccines or therapeutic drugs to control neosporosis in cattle.     

Trichinella spiralis infection induces β-actin co-localized with thymosin β4

A serological survey for Neospora caninum and Besnoitia besnoiti was carried out in beef and dairy cattle in South Australia. Serum samples of dairy cattle (n=133) from 9 properties and tank milk samples from a further 122 dairy herds were tested. An additional 810 sera from beef cattle from 51 properties were also tested. Testing at the individual animal level by IDEXX NEOSPORA X2 Ab test ELISA revealed a low prevalence of N. caninum antibodies of only 2.7% (95% CI; 1.6-3.7%) sera positive, as did the milk testing that showed 2.5% (95% CI; 1.4-3.6%) of tank milks being positive. At the herd level, 29.4% (95% CI; 16.9-41.9%) of beef, and 44.4% (95% CI; 12.0-76.9%) of dairy cattle herds showed serum antibodies. The highest within-herd prevalence in beef was 20% and 25%in dairy, which explains the low herd prevalence in dairy detected by bulk milk testing. Testing for B. besnoiti antibodies by PrioCHECK(?) Besnoitia Ab 2.0 ELISA initially identified 18.4% (95% CI: 15.8-21.0%) of 869 individual cattle sera as positive by ELISA at the manufacturer's suggested cut-off threshold (15 PP). Additional tests by immunoblot and IFAT, however, could not confirm any of the ELISA results. The use of a higher (40 PP) threshold in the ELISA is suggested to improve specificity. There is thus no evidence of B. besnoiti infection in South Australian cattle.     

Evaluation of three immunoserological techniques in the detection of porcine trichinellosis

Three immunoserological tests (IST) used for the detection of porcine trichinellosis, immunofluorescence (IF), enzyme-inmunoanalysis (EIA), and Western blot (WB), were compared. Three groups of animals were analyzed: Group 1, animals naturally infected with parasite burdens (PB) of <1 muscle larvae (ML)/g (n = 18); Group 2, animals naturally infected with PB of ≥2 ML/g (n = 23); Group 3, animals raised and home-slaughtered on farms in Argentina (n = 59). Animals from Groups 1 and 2 were identified in outbreaks and were analyzed by individual artificial digestion (AD) of ≥30 g of muscle. Animals in Group 3 were subjected to AD of 5 g of muscle.The detection percentages in sera of swine with the lower PB were 100% for IF, 72% for EIA, and 50% for WB. Eighty-three percent of the animals were serologically positive by two or three techniques. In pigs with the higher PB, the detection percentage was similar for IF and EIA (100% vs. 91%, respectively), and was lower for the WB (61%). Ninety-six percent of the animals were serologically positive by two or three techniques. Group 3 animals had similar detection percentages for the three techniques (IF, 30%; EIA, 29%; WB, 42%). Twenty-five percent of the animals were serologically positive by two or three techniques. Two animals were positive by AD with PB of 0.33 and 2.4 ML/g, and were positive for IF and WB, or IF, EIA, and WB. Results indicate that the sensitivity of each technique depends on the PB, and always ranked in sensitivity as IF > EIA > WB. For the lower PB, the decrease in the sensitivity is more pronounced for the EIA. Although the WB has a low sensitivity, the detection of the specific bands for Trichinella spiralis makes it a useful confirmatory tool. Considering that more than 83% of the parasitologically positive animals had 2 or 3 positive serological results using the techniques tested here, for the diagnosis of porcine trichinellosis, pigs positive by two of these serological techniques must be regarded as truly infected pigs.     

IgG antibodies from dourine infected horses identify a distinctive Trypanosoma equiperdum antigenic pattern of low molecular weight molecules

Diagnosis and control of dourine is strongly based on serological evidence, but knowledge of the humoral response of horses during infection is limited. In this study we developed a chemiluminescent immunoblotting (cIB) assay to characterise the Trypanosoma equiperdum antigen pattern recognised by IgGs from naturally or experimentally dourine-infected horses and analyse the kinetics of IgG humoral response following the infection. One compounding factor is that sera from uninfected animals often cross-react with T. equiperdum antigens. Development of the cIB assay was based on the hypothesis that serum IgGs from healthy and infected animals recognise different T. equiperdum antigen patterns. We used sera from 8 naturally infected horses which had recovered from Italian outbreaks and 2 experimentally infected mares. In addition, sera from 10 healthy control animals, eight of which were CFT positive but IFA negative for dourine, were collected from disease free regions. Sera were compared by the complement fixation test (CFT), indirect immune fluorescence (IFA) and the cIB assay.cIB analysis revealed that IgGs from infected horses, in contrast to IgGs from healhty horses, specifically recognise a T. equiperdum antigenic profile with low molecular weight bands ranging between 16 and 35 kDa. A time course experiment indicated that IgGs specific for the 16–35 kDa parasite protein fraction appear 17 days post-infection. The cIB assay confirmed all ten infected animals as positive and all controls as negative. This study demonstrated that analysis of IgGs by cIB can provide clear confirmation of trypanosome infection in horses, suggesting that this technique can be applied as a confirmatory serological test for dourine infection.     

Evidence for bovine besnoitiosis being endemic in Italy--first in vitro isolation of Besnoitia besnoiti from cattle born in Italy

Until 2009, bovine besnoitiosis had never been considered endemic in Italy and the only report on the disease in this country referred to animals imported from France shortly before. However, recently, an autochthonous outbreak of bovine besnoitiosis was reported in four herds located at the intersection of the borders between Emilia-Romagna, Toscana and Marche (Northern Apennine Mountains), which has led to an increased awareness concerning this disease. The present study describes a further outbreak of bovine besnoitiosis in Italy. The afflicted herd was a dairy herd with no evidence for contact with cattle from regions known to be endemic for bovine besnoitiosis. The farm investigation was initiated after a three-year old Holstein Friesian dairy cow with generalized thickening and lichenification of the skin was diagnosed with bovine besnoitiosis. The clinical diagnosis was confirmed by gross pathology, histopathology, serology and PCR. Bradyzoites released from tissue cysts obtained from the skin of this animal enabled the first in vitro isolation of Besnoitia besnoiti in Italy. This isolate was named Bb-Italy1. Sequencing of a 2118 bp spanning region including the complete internal transcribed spacer 1 and parts of the 18S and the 5.8S rRNA gene from DNA extracted from skin-derived zoites revealed a 99.9% identity to sequences known for other B. besnoiti isolated from cattle in Europe. Two GKO mice which had been inoculated intraperitoneally with bovine skin-derived bradyzoites became ill 7 days post inoculation. Parasitophorous vacuoles with multiplying zoites were observed in the cell culture inoculated with peritoneal fluids of these mice and a B. besnoiti infection in the mice and in the cell culture could be confirmed by real-time PCR. A serological investigation in the afflicted herd using immunoblots and an immunofluorescent antibody test (IFAT) revealed an overall herd seroprevalence of 9.7% (31/321), whereas within the female animals older than 2 years 17.0% (29/171) of the dams were tested positive. With one exception, an imported cow from Germany, all the seropositive animals were born in Italy. In connection with previously described autochthonous cases of bovine besnoitiosis the case described herein suggests that bovine besnoitiosis should be considered endemic in Italy.     

Development of an improved ESAT-6 and CFP-10 peptide-based cytokine flow cytometric assay for bovine tuberculosis

Control of bovine tuberculosis (bTB) continues to be a problem world-wide because of difficulties in identifying infected animals at all stages of infection. The use of the IFN-γ release assays (IGRA) as an ancillary test with the tuberculin skin tests has improved the ability to identify infected animals. However, infected animals may still be missed. The objective of the present study was to evaluate a rapid flow-cytometric assay based on intracellular cytokine staining as an alternative to the in vitro IFN-γ release assay (IGRA). Antigen-specific cells producing IFN-γ were identified after a 6 h stimulation with PPD-B, PPD-A and ESAT-6/CFP-10. Defined groups of animals naturally infected with Mycobacterium bovis (Mbv), animals infected with non-tuberculous mycobacteria (NTM), and uninfected control animals were analysed to evaluate the sensitivity and specificity of the optimized assay. Both antemortem and postmortem diagnostic tests were carried out to verify the status of infection. We show that IFN-γ is induced in T cells from whole blood samples from cattle infected with Mbv 6 h post stimulation with PPD-B, PPD-A and ESAT-6/CFP-10, whereas non-infected animals did not respond. Four colour flow cytometric analysis demonstrated responding cells were CD45R0⁺CD69⁺CD4⁺ memory T cells. Also, the response to stimulation with ESAT-6/CFP-10 can be used to distinguish between cattle infected with Mbv and cattle exposed to NTM. Although further studies are needed, the results indicate that detection of intracellular IFN-γ may represent an important alternative approach for improved method of detection of cattle secreting IFN-γ below levels of detection in culture medium.     

Oxidative stress in dairy cows seropositives for Neospora caninum

Bovine neosporosis is caused by the protozoan Neospora caninum and is one of the major causes of abortion in cows. Cattle are intermediate hosts of this parasite and may have asymptomatic or symptomatic infections. Therefore, the aim of this study was to evaluate oxidative stress marker reactive oxygen species (ROS), thiobarbituric reactive acid substances (TBARS) levels, glutathione S-transferase (GST), adenosine deaminase (ADA), and butyrylcholinesterase (BChE) activities in dairy cows seropositives for N. caninum (asymptomatic or symptomatic). Dairy cows (n = 90) were tested by immunofluorescent antibody assay (IFA) for N. caninum and divided accordingly into three groups: the group A (seronegatives, n = 30), the group B (seropositives and asymptomatic, n = 30), and the group C (seropositives and symptomatic, n = 30). It was observed increased levels of TBARS and reduced (P < 0.05) BChE activity in seropositives either asymptomatic or symptomatic animals. ROS levels and ADA activity increased, and GST activity decreased (P < 0.05) only in seropositives symptomatic dairy cows (the group C) compared to seronegatives dairy cows (the group A). Based on these results, it was observed that seropositive animals showed cell damage associated with oxidative stress and inflammation, mainly in those with symptomatic infections. Increased seric ROS levels and BChE activity may have influenced N. caninum pathogenesis in symptomatic animals due to increased cell damage and exacerbated inflammatory response, leading to the development of clinical signs.     

A longitudinal study of Besnoitia besnoiti infections and seasonal abundance of Stomoxys calcitrans in a dairy cattle farm of southwest France

Bovine besnoitiosis, caused by the cyst-forming apicomplexan Besnoitia besnoiti, is commonly reported in some restricted regions of South-Western Europe, and in larger regions of Africa and Asia. This infection is thought to be transmitted by blood feeding insects and is responsible for major economic losses in cattle production. A recent emergence in Europe, notified in the Centre of France, Spain and Germany, has attracted more attention to this disease. Clinical signs could appear in some animals; however, many infected cattle remain asymptomatic or show scleral-conjunctival cysts (SCC) only. Recent development of serological methods allows carrying out seroepidemiological field studies. In this respect, a long-term investigation was performed in a dairy cattle farm localized in an enzootic area of besnoitiosis of South-western France between March 2008 and May 2009. The objective was to estimate the seasonal pattern of B. besnoiti infections based on the presence of SCC and serology (ELISA and Western blot). In parallel, an entomological survey was conducted to describe population dynamics of Stomoxys calcitrans and Tabanidae species. The seroprevalence determined by Western blot in a cohort of 57 animals continuously present during the whole survey increased from 30% in March 2008 to 89.5% in May 2009 and was always higher than the prevalence based on clinically assessed SCC. New positive B. besnoitia seroconversions occurred throughout the year with the highest number in spring. In addition, many seroconversions were reported in the two months before turn-out and could be associated with a high indoors activity of S. calcitrans during this period.     

Diagnosis of canine brucellosis by ELISA using an antigen obtained from wild Brucella canis

An indirect ELISA test was developed for the diagnosis of Brucella canis infection in dogs. A bacterial whole cell extract was used as a solid phase antigen, using B. canis isolated from an infected animal. Sera from culture-positive and healthy negative animals were used as internal reference controls. The cut-off point was determined by a mathematical formula for a statistically valid value, which defined the upper prediction limit, based on the upper tail of the t-distribution of 21 negative control sera readings, for the confidence level of 99.5%. The sensitivity and specificity of the ELISA test were 95% and 91%, respectively. The ELISA test showed a significant concordance index (K = 0.84) with the agar gel immunodiffusion test. The reliability of the ELISA for the detection of infected animals was established by a double blind study testing 280 sera provided by serum banks from different diagnostic and research institutions and analyzed by ROC Curve.     

Evaluation of long-term antibody responses to two inactivated bovine viral diarrhoea virus (BVDV) vaccines

The aim of the present study was to determine the serological response of heifers after vaccination with two inactivated bovine viral diarrhoea virus (BVDV) vaccines by means of various ELISA tests. Three dairy farms were selected from the Galicia region of Spain. In each herd, a batch of heifers to be vaccinated for the first time was selected and followed for 15 months. Heifers from farm 1 (n = 25) were vaccinated with Vaccine A, whereas heifers from farm 2 (n = 16) were vaccinated with Vaccine B. Heifers from farm 3 (n = 17), where no BVDV vaccines were used, acted as controls. Blood samples were analyzed periodically for BVDV antibodies, using five commercial ELISAs, based on BVDV p80 antigen or whole virus.At the end of the study, none of the animals vaccinated with Vaccine A seroconverted according to p80 antibody status, whereas up to 80% tested positive by ELISA against whole virus antigen. For the animals vaccinated with Vaccine B, 2/16 animals seroconverted according to p80 antibody ELISAs, whereas all had seroconverted according to the ELISA against whole virus antigen. In most cases, based on the use of ELISAs to detect specific antibodies against the p80 protein, at 15 months post-vaccination with inactivated BVDV vaccines the responses did not seem to interfere with detection of antibody to BVDV infection. However, the finding of a small proportion of vaccinated animals seropositive against BVDV p80 antigen suggests that antibodies that interfere with diagnosis of BVDV infection within the herd could exist, even when using p80 ELISAs.     

Bovine besnoitiosis in Germany

This paper reports a case of natural occurring bovine besnoitiosis in Germany. The skin lesions consisted of multifocal hypotrichosis and alopecia, lichenification, erythema and seborrhoea. Histopathologic findings revealed characteristic cysts of Besnoitia spp. The diagnosis was confirmed by serology and the species Besnoitia besnoiti was identified by polymerase chain reaction (PCR).