Biochemical and molecular characterization of Campylobacter fetus isolates from bulls subjected to bovine genital campylobacteriosis diagnosis in Spain.

BACKGROUND: Bovine genital campylobacteriosis (BGC) is caused by Campylobacter fetus subsp. venerealis (Cfv) including its biovar intermedius (Cfvi). This sexually transmitted disease induces early reproductive failure causing considerable economic losses in the cattle industry. Using a collection of well-characterized isolates (n = 13), C. fetus field isolates (n = 64) and saprophytic isolates resembling Campylobacter (n = 75) obtained from smegma samples of breeding bulls, this study evaluated the concordance of the most used phenotypic (H2S production in cysteine medium and 1% glycine tolerance) and molecular (PCR) methods for the diagnosis of BGC and assessed possible cross-reactions in the molecular diagnostic methods. RESULTS: Characterization at the subspecies level (fetus vs. venerealis) of C. fetus isolated from bull preputial samples using phenotypic and molecular (PCR targeting nahE and ISCfe1) methods showed moderate concordance (κ = 0.462; CI: 0.256-0.669). No cross-reactions were observed with other saprophytic microaerophilic species or with other Campylobacter species that can be present in preputial samples. Whole genome sequencing (WGS) of discrepant isolates showed 100% agreement with PCR identification. For the differentiation of Cfv biovars, comparison of the H2S test (at 72 h and 5 days of incubation) and a PCR targeting the L-cysteine transporter genes showed higher concordance when H2S production was assessed after 5 days (72 h; κ = 0.553, 0.329-0.778 CI vs. 5 days; κ = 0.881, 0.631-1 CI), evidencing the efficacy of a longer incubation time. CONCLUSIONS: This study confirmed the limitations of biochemical tests to correctly identify C. fetus subspecies and biovars. However, in the case of biovars, when extended incubation times for the H2S test (5 days) were used, phenotypic identification results were significantly improved, although PCR-based methods produced more accurate results. Perfect agreement of WGS with the PCR results and absence of cross-reactions with non-C. fetus saprophytic bacteria from the smegma demonstrated the usefulness of these methods. Nevertheless, the identification of new C. fetus subspecies-specific genes would help to improve BGC diagnosis.

Seroprevalence of influenza D virus in bulls in Argentina.

Influenza D virus (IDV) is considered a new agent involved in bovine respiratory disease (BRD). Based on seroprevalence studies or isolation from clinical samples, this virus has been detected on several continents and in several animal species, including cattle, pigs, camel, horses, and goats. We used an indirect in-house ELISA to detect anti-IDV antibodies in 165 serum samples from bulls on 116 farms in the province of La Pampa, Argentina. Eighty-five of 116 (73%) farms had at least 1 positive animal, and 112 of 165 (68%) of the analyzed samples were positive. There were no significant differences in the proportion of seropositive samples depending on the geographic region in which the samples were taken. Our results suggest that IDV infection is endemic in La Pampa; the clinical importance of IDV in Argentina remains to be investigated.

Multilocus analysis reveals further genetic differences between Tritrichomonas foetus from cats and cattle.

Tritrichomonas foetus isolates from feline and bovine origin has been previously shown to carry a certain degree of genetic heterogeneity. Here, novel candidate molecular markers were developed by means of multilocus sequence typing of the gap2 gene (encoding for T. foetus glyceraldehyde-3-phosphate dehydrogenase), ITS region, the TR7/TR8 variable-length repeat and microsatellite genotyping. These markers were used to characterize T. foetus field isolates from bulls and domestic cats and to compare phylogenetically with the following ATCC isolates: T. foetus isolated from cattle and pig (syn. Tritrichomonas suis), Tritrichomonas mobilensis, Tetratrichomonas gallinarum and Pentatrichomonas hominis. Among them, TFMS10 and TFMS7 were found to be the most polymorphic markers. Moreover, an 809 bp fragment of the gap2 gene was successfully amplified from all the trichomonads included in this study and the sequence analysis revealed differences between T. foetus porcine and feline genotypes and T. mobilensis in comparison to the bovine T. foetus ATCC isolate. The TR7/TR8 repeat pattern was not reproducible, being only consistent the fragments of approximately 110 and 217 bp. Sequence analysis of the latter revealed the existence of 3 SNPs resulting in 98.6 % homology between bovine and feline isolates. A search for similar sequences was carried out to develop a Restriction Length Fragment Polymorphism analysis. A 503 bp region, named TF1, revealed the existence of two BbvI restriction enzyme sites that were able to generate different length fragments for T. foetus feline and bovine isolates. Finally, the neighbour-joining analyses showed that T. foetus porcine genotype clusters together with bovine genotype, whereas T. mobilensis and the feline genotype form a separate cluster.

Detección de Chlamydia abortus en pérdidas reproductivas de bovinos en la provincia de La Pampa, Argentina / Detection of Chlamydia abortus in bovine reproductive losses in the province of La Pampa, Argentina

Las pérdidas reproductivas constituyen una causa importante de pérdida económica en el ganado bovino, aunque en más del 50% de los casos la etiología es desconocida. Las especies de la familia Chlamydiaceae han sido asociadas con abortos en bovinos y otras espeChlamydia abortus; cies animales, pero no existen datos al respecto en la República Argentina. El objetivo de este trabajo fue determinar la presencia de Chlamydia spp. y de Chlamydia abortus en pérdidas reproductivas de ganado bovino en La Pampa, Argentina. Se estudiaron 251 muestras provenientes de abortos y mortinatos. Se realizó PCR en tiempo real para la detección de la familia Chlamydiaceae y ArrayTube para la identificación de las especies presentes. Se detectó ADN de la familia Chlamydiaceae en 12 muestras (4,78%); el 83,33% (10/12) correspondió a abortos y el 16,66% (2/12) a mortinatos. El análisis por ArrayTube detectó C. abortus en 5 muestras (1,99% del total, 41,67% de las muestras con detección de Chlamydiaceae). Este trabajo presenta la primera confirmación de la presencia de ADN de diversas especies de Chlamydiaceae (incluida C. abortus) en muestras de pérdidas reproductivas de ganado bovino en Argentina. El valor de prevalencia hallado (4,78%) debe ser tomado como un valor basal, debido al tipo de muestras estudiadas. Se halló material genético de Chlamydiaceae que no coincidió con ninguna de las especies conocidas; esto podría deberse a variantes intraespecie o a especies autóctonas aún no descriptas. Es necesario avanzar en el estudio de la infección por estas bacterias en el ganado bovino de Argentina para conocer su dimensión y analizar su impacto económico y zoonótico, y también para planear medidas de prevención y control.

Reproductive losses linked to an infectious etiology in bovine cattle are a major economic concern worldwide. In Argentina, more than 50% of abortion cases have unknown causes. Species belonging to Chlamydiaceae family are frequent etiologic agents of abortion around the world; however, there is yet no information on their prevalence in Argentina. The objective of this work was to identify Chlamydia spp., and particularly C. abortus in reproductive losses from bovine cattle in La Pampa, Argentina. Real time PCR targeting Chlamydiaceae-specific DNA fragments was performed on 251 samples obtained from bovine abortions and stillborns, and ArrayTube was used for species identification on positive samples. Chlamydiaceae DNA was detected in 12 samples of aborted fetuses (4.78%), 83.33% (10/12) accounting for abortions and 16.66% (2/12) for stillborns. C. abortus was detected by ArrayTube in 5 cases (1.99% of all samples, and 41.67% of Chlamydiaceae positive samples). This study shows the first detection of Chlamydiaceae and C. abortus DNA on reproductive losses of bovine cattle in Argentina, and the described prevalence value (4.78%) should be taken as baseline value due to the type of samples analyzed. Detection of genetic material from Chlamydiaceae not matching any of the studied species could be due to intraspecies variants or local species not yet described. Further research on Chlamydia infections in bovine cattle in Argentina is imperative to describe their range, to analyze their economic and zoonotic implications and to make recommendations about prevention and control measures.

La técnica de polarización fluorescente para el diagnóstico de Brucella en Chaetophractus villosus / Technique of fluorescence polarization for the diagnosis of Brucella in Chaetophractus villosus / A técnica de polarização fluorescente para o diagnóstico de Brucella em Chaetophractus villosus

La brucelosis es una enfermedad muy extendida por todo el mundo. El diagnóstico se realiza mediante el uso de diferentes técnicas, entre ellas el aislamiento del agente etiológico o por serología detectando anticuerpos específicos. Todos estos métodos están estandarizados y validados en general para las distintas especies animales de interés zootécnico. En animales silvestres pueden utilizarse los mismos procedimientos serológicos, pero cada uno debe ser validado para la especie animal estudiada. El objetivo de este trabajo fue comprobar si la técnica de Polarización Fluorescente (FPA) utilizada para la determinación de anticuerpos contra Brucella en bovinos puede ser usada para el diagnóstico de Brucella en el armadillo (Chaetophractus villosus). Para ello se tomaron 150 muestras de sangre de Chaetophractus villosus. Veinticuatro muestras fueron positivas por medio de las técnicas de antígeno bufferado en placa, seroaglutinación lenta en tubo, 2-mercaptoetanol y fijación del complemento. Las mismas muestras resultaron positivas a la técnica de Polarización Fluorescente, estableciéndose un límite de corte de 82 mP. El índice Kappa registrado fue de 1 para todos los tests de diagnóstico comparados con FPA (IC: 0,84-1).

Brucellosis is a disease widespread throughout the world. The diagnosis is made through the use of different techniques including the isolation of the etiological agent or by serology detecting specific antibodies. All these methods are standardized and validated in general for animal species of zoo-technical interest. In wild animals, serological procedures may be used, but each should be validated for the particular animal species studied. The objective of this work was to verify if the technique of Fluorescent Polarization used for the determination of antibodies against Brucella in ruminants can be used for the diagnosis of Brucella in Chaetophractus villosus. To this aim, s 150 blood samples of Chaetophractus villosus were taken. Twenty-four samples were positive by the techniques of Antigen Buffered in Plate, serum agglutination test (SAT), 2-mercaptoethanol (2-ME) agglutination test and complement fixation test (CFT). The same samples were also positive by Fluorescent Polarization, and a cut-off limit of 82 mP was established. The KAPPA index was 1 for all diagnostic tests compared to FPA (CI: 0.84-1).

A brucelose é uma doença muito espalhada pelo mundo. O diagnóstico é feito por meio de diferentes técnicas, incluindo o isolamento do agente etiológico ou por sorologia detectando anticorpos específicos. Todos esses métodos são padronizados e validados em geral para diferentes espécies animais de interesse zootécnico. Em animais silvestres podem ser utilizados os mesmos métodos serológicos, mas cada um tem de ser validado para a espécie de animal estudada. O objectivo deste estudo foi testar se a técnica de Polarização Fluorescente utilizada para a determinação de anticorpos contra Brucella em bovinos pode ser utilizada para o diagnóstico de Brucella em tatu (Chaetophractus villosus). Para isso, 150 amostras de sangue de Chaetophractus villosus foram coletadas. Vinte e quatro amostras foram positivas por técnicas de antígeno bufferado em placa, soroaglutinação lenta em tubo, 2-mercaptoetanol e fixação do complemento. As mesmas amostras foram positivas para a técnica de Polarização Fluorescente, estabelecendo um limite de corte de 82 mP. O índice Kappa registrado foi de 1 para todos os testes de diagnóstico em comparação com FPA (CI: 0,84-1).

[Detection of Chlamydia abortus in bovine reproductive losses in the province of La Pampa, Argentina]. / Detección de Chlamydia abortus en pérdidas reproductivas de bovinos en la provincia de La Pampa, Argentina.

Reproductive losses linked to an infectious etiology in bovine cattle are a major economic concern worldwide. In Argentina, more than 50% of abortion cases have unknown causes. Species belonging to Chlamydiaceae family are frequent etiologic agents of abortion around the world; however, there is yet no information on their prevalence in Argentina. The objective of this work was to identify Chlamydia spp., and particularly C. abortus in reproductive losses from bovine cattle in La Pampa, Argentina. Real time PCR targeting Chlamydiaceae-specific DNA fragments was performed on 251 samples obtained from bovine abortions and stillborns, and ArrayTube was used for species identification on positive samples. Chlamydiaceae DNA was detected in 12 samples of aborted fetuses (4.78%), 83.33% (10/12) accounting for abortions and 16.66% (2/12) for stillborns. C. abortus was detected by ArrayTube in 5 cases (1.99% of all samples, and 41.67% of Chlamydiaceae positive samples). This study shows the first detection of Chlamydiaceae and C. abortus DNA on reproductive losses of bovine cattle in Argentina, and the described prevalence value (4.78%) should be taken as baseline value due to the type of samples analyzed. Detection of genetic material from Chlamydiaceae not matching any of the studied species could be due to intraspecies variants or local species not yet described. Further research on Chlamydia infections in bovine cattle in Argentina is imperative to describe their range, to analyze their economic and zoonotic implications and to make recommendations about prevention and control measures.

An Ibero-American inter-laboratory trial to evaluate serological tests for the detection of anti-Neospora caninum antibodies in cattle.

We carried out an inter-laboratory trial to compare the serological tests commonly used for the detection of specific Neospora caninum antibodies in cattle in Ibero-American countries. A total of eight laboratories participated from the following countries: Argentina (n = 4), Brazil (n = 1), Peru (n = 1), Mexico (n = 1), and Spain (n = 1). A blind panel of well-characterized cattle sera (n = 143) and sera representative of the target population (n = 351) was tested by seven in-house indirect fluorescent antibody tests (IFATs 1-7) and three enzyme-linked immunosorbent assays (ELISAs 1-3; two in-house and one commercial). Diagnostic performance of the serological tests was calculated and compared according to the following criteria: (1) the "Pre-test information," which uses previous epidemiological and serological data; (2) the "Majority of tests," which classifies a serum as positive or negative according to the results obtained by most tests evaluated. Unexpectedly, six tests showed either sensitivity (Se) or specificity (Sp) values lower than 90%. In contrast, the best tests in terms of Se, Sp, and area under the ROC curve (AUC) values were IFAT 1 and optimized ELISA 1 and ELISA 2. We evaluated a high number of IFATs, which are the most widely used tests in Ibero-America. The significant discordances observed among the tests regardless of the criteria employed hinder control programs and urge the use of a common test or with similar performances to either the optimized IFAT 1 and ELISAs 1 and 2.

Clearance of Tritrichomonas foetus in experimentally infected heifers protected with vaccines based on killed-T. foetus with different adjuvants.

Tritrichomonas foetus is a flagellated protozoan that causes a sexually transmitted disease in cattle. Trichomonosis is characterized by early abortions, subfertility and a significant decrease in productivity. Vaccine preparations containing whole T. foetus can reduce the time of residence of the pathogen in the host cervix after experimental infection. Here, T. foetus vaccines prepared with different adjuvants were tested, in parallel with a commercial vaccine, for their efficacy to clear the infection. The median time for clearance of infection was 69days in non-immunized animals, 55days in animals treated with aluminum hydroxide, 41days with oil-in-water or saponin based vaccines or with a commercial vaccine and 27days in animals treated with saponin plus aluminum hydroxide. A slight increase in the risk of T. foetus clearance from the genital tract was found with the saponin based vaccine (hazard ratio, 2.52; 95% confidence interval, 1.03-6.17) or the commercial vaccine (hazard ratio, 2.61; 95% confidence interval, 1.07-6.38). A significant increase in the risk of T. foetus clearance was found with the combination of saponin plus aluminum hydroxide based vaccine (hazard ratio, 5.12; 95% confidence interval, 2.04-12.83).

Seroepidemiological study of Neospora caninum in beef and dairy cattle in La Pampa, Argentina.

Neospora caninum is considered one of the major causes of abortion in cattle. The aim of this study was to examine and quantify the extent of the infection in cattle in a representative region of Argentina (La Pampa, province). An average sample size of 36 sera per herd was selected from 97 beef and 24 dairy herds. A total of 4334 serum samples were tested for specific anti- Neospora caninum IgG using an indirect-ELISA and 302 seropositive-ELISA sera were re-examined using an Avidity-ELISA procedure for N.caninum. The overall estimated seroprevalence for N.caninun was 9.6% (95%CI: 8.7%; 10.5%). Levels of seroprevalence were significantly different in beef 7.0% and dairy 20.3% cattle. Disease distribution seems to be associated with climatic conditions as well as the management system. Cows in the east and central regions were at a 4.5-fold and 2.0-fold higher risk, respectively, of being N. caninum seropositive compared with cows in west region. Levels of recent infection were evaluated through an avidity ELISA in seropositive animals, being registered a 0.56% and a 1.71% of recent infection in beef and dairy cattle respectively (p = 0.006). The results revealed that dairy cows had 3.1(95%CI: 1.4; 7.0) higher risk of contracting Neoporosis through horizontal transmission than beef cows. A relationship between Brucella abortus and N. caninum seroprevalence was also observed. The risk of being N. caninum seropositive was two times higher where Brucellosis seroprevalence was >3.5%. These results reveal the distribution of N. caninum infection in the cattle population in La Pampa, Argentina.

Presence of antibodies against Leptospira serovars in Chaetophractus villosus (Mammalia, Dasypodidae), La Pampa province, Argentina.

Leptospirosis is a zoonosis of worldwide distribution. The aim of this study was to examine the presence of antibodies against 21 Leptospira reactive serovars in Chaetophractus villosus in La Pampa province, Argentina, using the microscopic agglutination test (MAT). Pathologic changes compatible with leptospirosis and in situ detection of the agent by immunohistochemistry were studied in 24 and 3 individuals respectively. Only 35/150 (23.3%) serum samples had antibodies against Leptospira sp. Six percent of the samples reacted with serovar Canicola, 4.7% with serovar Castellonis, 1.3% with serovar Icterohemorrhagieae and 0.7% with serovar Hardjo. Sixteen (10.6%) serum samples agglutinated with Castellonis-Icterohemorrhagiae and Canicola-Castellonis serovars, both with 4.7%, and Canicola-Hardjo and Castellonis-Canicola-Icterohemorrhagiae both with 0.6%. Fourteen animals had variable degrees of lesions, which were more severe in animals with higher serological titers (3200), and Leptospira sp. was detected in 3 animals by immunohistochemistry. These results represent the first record of the presence of Leptospira in C. villosus in La Pampa.

Presencia de anticuerpos contra serovares de Leptospira en Chaetophractus villosus (Mammalia, Dasypodidae), provincia de La Pampa, Argentina / Presence of antibodies against Leptospira serovars in Chaetophractus villosus (Mammalia, Dasypodidae), La Pampa province, Argentina

Leptospirosis is a zoonosis of worldwide distribution. The aim of this study was to examine the presence of antibodies against 21 Leptospira reactive serovars in Chaetophractus villosus in La Pampa province, Argentina, using the microscopic agglutination test (MAT). Pathologic changes compatible with leptospirosis and in situ detection of the agent by immunohistochemistry were studied in 24 and 3 individuals respectively. Only 35/150 (23.3%) serum samples had antibodies against Leptospira sp. Six percent of the samples reacted with serovar Canicola, 4.7% with serovar Castellonis, 1.3% with serovar Icterohemorrhagieae and 0.7% with serovar Hardjo. Sixteen (10.6%) serum samples agglutinated with Castellonis­Icterohemorrhagiae and Canicola­Castellonis serovars, both with 4.7%, and Canicola­Hardjo and Castellonis­Canicola­Icterohemorrhagiae both with 0.6%. Fourteen animals had variable degrees of lesions, which were more severe in animals with higher serological titers (3200), and Leptospira sp. was detected in 3 animals by immunohistochemistry. These results represent the first record of the presence of Leptospira in C. villosus in La Pamp

La leptospirosis es una zoonosis de distribución mundial. Nuestro objetivo fue examinar la presencia de anticuerpos contra 21 serovares reactivos de Leptospira en Chaetopractus villosus en la provincia de La Pampa, Argentina, mediante la prueba de aglutinación microscópica (MAT). Se realizó el estudio histopatológico y la detección in situ del agente por inmunohistoquímica en 24 y 3 individuos, respectivamente. Solo 35/150 (23,3%) muestras de suero presentaron anticuerpos contra Leptospira sp. Seis por ciento reaccionaron al serovar Canicola; 4,7% a Castellonis; 1,3% a Icterohemorrhagieae y 0,7% a Hardjo. Dieciséis (10,6%) sueros aglutinaron con Canicola-Castellonis y Castellonis-Icterohemorrhagiae, ambos con 4,7%, y con Canicola-Hardjo y Castellonis-Canicola-Icterohemorrhagiae, ambos con 0,6%. En 14 animales se encontraron lesiones compatibles, las que resultaron más graves en animales con títulos serológicos elevados (3200). En 3 animales estudiados se detectó el agente causal por inmunohistoquímica. Estos resultados constituyen los primeros registros de la presencia de Leptospira en C. villosus en La Pampa

Presence of antibodies against Leptospira serovars in Chaetophractus villosus (Mammalia, Dasypodidae), La Pampa province, Argentina. / Presence of antibodies against Leptospira serovars in Chaetophractus villosus (Mammalia, Dasypodidae), La Pampa province, Argentina.

Leptospirosis is a zoonosis of worldwide distribution. The aim of this study was to examine the presence of antibodies against 21 Leptospira reactive serovars in Chaetophractus villosus in La Pampa province, Argentina, using the microscopic agglutination test (MAT). Pathologic changes compatible with leptospirosis and in situ detection of the agent by immunohistochemistry were studied in 24 and 3 individuals respectively. Only 35/150 (23.3

) serum samples had antibodies against Leptospira sp. Six percent of the samples reacted with serovar Canicola, 4.7

with serovar Castellonis, 1.3

with serovar Icterohemorrhagieae and 0.7

with serovar Hardjo. Sixteen (10.6

) serum samples agglutinated with Castellonis-Icterohemorrhagiae and Canicola-Castellonis serovars, both with 4.7

, and Canicola-Hardjo and Castellonis-Canicola-Icterohemorrhagiae both with 0.6

. Fourteen animals had variable degrees of lesions, which were more severe in animals with higher serological titers (3200), and Leptospira sp. was detected in 3 animals by immunohistochemistry. These results represent the first record of the presence of Leptospira in C. villosus in La Pampa.

Brucella suis in armadillos (Chaetophractus villosus) from La Pampa, Argentina.

Brucellosis is a zoonotic disease transmitted from an animal reservoir to humans. Both, wildlife and domestic animals, contribute to the spreading of these zoonosis. The surveillance of the animal health status is strictly regulated for domestic animals, whereas disease monitoring in wildlife does not exist. The aim of the present study was to provide data on the prevalence of anti-Brucella antibodies in Chaetophractus villosus from a region of La Pampa, Argentina to assess public health risks. The C. villosus is endemic to South America, and in Argentina it represents a food resource for human consumption. A total of 150 sera of armadillos bleeding between 2007 and 2010 were tested using buffered plate antigen test (BPAT), serum agglutination test (SAT), 2-mercaptoethanol (2-ME) and complement fixation test (CFT), for the detection of anti-Brucella antibodies. Antibodies to Brucella sp. were found in 16% (24:150) of the armadillos tested using the BPAT test. All 24 positive samples were confirmed by the SAT, 2-ME and CFT tests. Strain isolation was attempted from liver and spleen samples of two animals with positive serology. Isolates were characterized by conventional biotyping and identification of specific DNA using polymerase chain reaction (PCR). A total of 2 isolates were recovered from spleen and liver. Both of them were identified as Brucella suis biovar 1. This preliminary study provides the first report on the seroprevalence of brucellosis and describes the first isolate of B. suis biovar 1 in C. villosus in Argentina.

First record of Toxoplasma gondii in Chaetophractus villosus in Argentina.

Toxoplasma gondii is an obligate intracellular apicomplexan parasite that causes abortion and reproductive disorder in domestic animals. T. gondii is a common worldwide disease in homeothermic animals, including birds and humans. The aim of the present study was to determine the presence of antibodies against T. gondii in the armadillo Chaetophractus villosus in the province of La Pampa, Argentina. Serum samples were collected from 150 individuals (70 males and 80 females). For serological detection of T. gondii, a latex agglutination test was first performed and then positive sera were confirmed with an indirect hemagglutination test, using 1:4 to 1:64 dilutions. Results showed that 27% (41) of the samples presented titers for antibodies against T. gondii. There were not significant differences between the presence of antibodies against T. gondii and age or sexes of the armadillos. Results show that presence of T. gondii antibodies in armadillos were associated with presence of pigs, and sheep, however there was not association with chickens and dairy cattle in capture site. T. gondii has an important presence in C. villosus population, suggesting a potential zoonotic risk for humans and wildlife animals when C. villosus meats are consumed raw or undercooked. This is the first record of the presence of antibodies against T. gondii in C. villosus.

Loop mediated isothermal amplification of 5.8S rDNA for specific detection of Tritrichomonas foetus.

Tritrichomonas foetus is the causative agent of bovine trichomonosis, a sexually transmitted disease leading to infertility and abortion. A test based on loop mediated isothermal amplification (LAMP) targeting the 5.8S rDNA subunit was designed for the specific identification of T. foetus. The LAMP assay was validated using 28 T. foetus and 35 non-T. foetus trichomonads strains. It did not exhibit cross-reaction with closely related parasites commonly found in smegma cultures like Tetratrichomonas spp. and Pentatrichomonas hominis. Bovine smegma did not show interferences for the detection of the parasite and, the sensitivity of the method (4×10(3) CFU/mL, approximately 10 cells/reaction) was slightly higher than that found for PCR amplification with TFR3 and TFR4 primers. The LAMP approach has potential applications for diagnosis and control of T. foetus and, practical use for low skill operators in rural areas.

Experimental infection of rabbits (Oryctolagus cuniculus) with Brucella suis biovar 1 isolated from wild hares (Lepus europaeus).

Brucella suis biovar 1 is the causative agent of brucellosis in several domestic and wild animals and it is a common agent of human brucellosis. European hares (Lepus europaeus) have been shown to be infected by B. suis biovar 1 and the transmission to other animals has been suggested. In this work, experimental rabbits (Cuniculus orictolagus) were infected with B. suis biovar 1 isolated from wild hares. Infected rabbits showed high serological response in 2 weeks after discharge and typical granulomatous lesions (2mm diameter) were found in liver, spleen and kidneys after 50 days. B. suis biovar 1 was cultured from the lesion of the organs mentioned above as well as from urine, placenta and fetuses. These data suggest that hares are a potential source for horizontal transmission of B. suis biovar 1 to other mammalians.

Tritrichomonas foetus isolates from cats and cattle show minor genetic differences in unrelated loci ITS-2 and EF-1α.

The protozoan parasite Tritrichomonas foetus is well known as an important causative agent of infertility and abortion in cattle (bovine trichomonosis). This World Organisation for Animal Health (O.I.E.) notifiable disease is thought to be under control in many countries including Switzerland. In recent studies, however, T. foetus has also been identified as an intestinal parasite that causes chronic large-bowel diarrhoea in cats. Since the feline isolates were considered indistinguishable from bovine isolates, the possibility and risk of parasite transmission from cats to cattle and vice versa has been intensively discussed in current literature. Therefore, we investigated if cat and cattle isolates are genetically distinct from each other or in fact represent identical genotypes. For this purpose, two independent genetic loci were selected that turned out to be well-suited for a PCR sequencing-based genotyping of trichomonad isolates: (i) previously published internal transcribed spacer region 2 (ITS-2) and (ii) a semi-conserved sequence stretch of the elongation factor-1 alpha (EF-1α) gene used for the first time in the present study. Respective comparative analyses revealed that both loci were sufficiently variable to allow unambiguous genetic discrimination between different trichomonad species. Comparison of both genetic loci confirmed that T. suis and T. mobilensis are phylogenetically very close to T. foetus. Moreover, these two genetic markers were suited to define host-specific genotypes of T. foetus. Both loci showed single base differences between cat and cattle isolates but showed full sequence identity within strains from either cat or cattle isolates. Furthermore, an additional PCR with a forward primer designed to specifically amplify the bovine sequence of EF-1α was able to discriminate bovine isolates of T. foetus from feline isolates and also from other trichomonads. The implications these minor genetic differences may have on the biological properties of the distinct isolates remain to be investigated.

An applied printing immunoassay with recombinant Nc-SAG1 for detection of antibodies to Neospora caninum in cattle.

Neospora caninum is a protozoan parasite that causes an important reproductive disease in cattle. Neospora caninum surface antigen 1 (Nc-SAG1) is an immunodominant candidate for the development of a diagnostic reagent for neosporosis. The current study describes the development and evaluation of an antigen print immunoassay (APIA) with recombinant Nc-SAG1 for the detection of specific antibodies to N. caninum in cattle. The concordance between APIA and a commercial enzyme-linked immunosorbent assay (ELISA) was evaluated with 232 serum samples from experimentally and naturally infected cattle. Sixty-one (26.7%) samples were positive for antibodies to N. caninum by ELISA and 58 (25.4%) by APIA. The new assay had a sensitivity of 85% and a specificity of 96%. These results, along with the potential of APIA to evolve into a multiple antigen detection format, suggest that this method would be a reliable diagnostic test for detection of antibodies to N. caninum in cattle.

Neosporosis y toxoplasmosis en la liebre europea (Lepus europaeus) en la provincia de La Pampa (Argentina) / Infectious diseases and reproductive status in european hare (Lepus europaeus) in La Pampa province (Argentina)

El objetivo de este trabajo fue determinar la prevalencia a Neospora caninum (Nc) y Toxoplasma gondii (Tg) y conocer el periodo reproductivo de la liebre europea (Lepus europaeus) en la provincia de La Pampa (Argentina). Para ello se muestrearon 106 liebres procedentes de acopiadores de la provincia de La Pampa. Los sueros fueron analizados por un ensayo inmunoenzimático de competición para determinar anticuerpos a Nc y para Tg se realizó una prueba de hemaglutinación indirecta. Se encontraron 66 hembras, de las cuales 13 estaban preñadas, y 40 machos (p=0,01). Sobre un total de 44 sueros analizados para Nc, 5 (11,4%) dieron positivo. De los 106 sueros estudiados para Tg ninguno fue positivo.

The objective of this study was to determine the prevalence of these diseases and to know the reproductive season of the European hare (Lepus europaeus) in the Province of La Pampa (Argentina). To that aim, 106 hares coming from different places from La Pampa province were sampled. .Nc antibody diagnosis was performed by means of a competitive enzimoimmunoassay (ELISA-c), and an hemo-agglutination indirect assay (HAI) for Tg were carried out to know the disease status. From the total of hares sampled, 40 were male, 66 female (p=0.01) and 13 (19.7%) of them were detected as pregnant. Nc sero-prevalence was 11.4% over a total of 44. From 106 sera analyzed by HAI no one was positive for Tg.