Positive cases of bovine genital campylobacteriosis and bovine trichomonosis in the eradication program of La Pampa, Argentina: 2008-2021.

Bovine genital campylobacteriosis, caused by the gram-negative bacteria Campylobacter fetus venerealis, and bovine trichomonosis, caused by the parasite protozoan Tritrichomonas foetus, are venereal diseases that occur with long intercalving periods and abortion. The control of both diseases relies on microbiological testing and culling infected bulls. Vaccination and antibiotic treatment may help in controlling campylobacteriosis but are not recommended for trichomonosis control. Several regions of the world have active control programs for trichomonosis, not campylobacteriosis. In Argentina, the state of La Pampa aims to eradicate trichomonosis and campylobacteriosis by imposing annual diagnostic testing of every bull and slaughtering positive animals. Prior studies indicated a declining trend in the prevalence of campylobacteriosis and trichomonosis in La Pampa. It was also proposed that the prevalence of one disease could be estimated from the prevalence of the other. The purpose of this retrospective analysis of data gathered from 2008 to 2021 was to determine the La Pampa program's efficacy. Descriptive statistics were employed to determine the reason behind the correlation between tricomonosis and campylobacteriosis diagnostic results. The outcomes refute the notion that this program of venereal eradication was a success. Furthermore, an excess of false positives in both diagnoses may have contributed to the correlation between the prevalences of campylobactriosis and trichomonosis. The practice of killing animals without verifying positive results hinders the determination of disease prevalence and results in the death of numerous healthy animals.

In vitro efficacy testing of ivermectin against Tritrichomonas foetus trophozoites.

There are no recommended drugs to treat cattle infected with the protozoan Tritrichomonas foetus (TF). Ivermectin, widely used in the treatment of intestinal parasites, was found effective against some protozoa growing in vitro. Here, its effectiveness against a TF line was investigated. Trophozoites were incubated in media with increasing concentrations of ivermectin and mortality was determined after 24 h. Ivermectin killed cells with a mean maximum effective concentration (EC50) of 2.47 µg mL-1. The effective concentration of ivermectin was rather high for a formulation suitable for systemic treatment. However, topical treatment of animals against TF could still be considered and tested.

In vitro culture of Tritrichomonas foetus in bovine cervico-vaginal fluid.

Tritrichomonas foetus is the causative agent of bovine trichomonosis, a venereal disease that can lead to fetal loss. T. foetus proliferates in the vagina and cervix and invades the uterus and fetal cavities. It is not clear how T. foetus grows in the host or how its infection rarely demonstrable after 4 months, is controlled. Cervical vaginal mucus (CVM) is a protective barrier against potentially harmful microorganisms. Here, we demonstrate that bovine CVM is a medium in which this protozoan parasite can grow in vitro. T. foetus multiplied at different rates depending on the time of the estrous period from which the CVM was obtained. Growth rates were higher in CVM obtained 7-10 days after estrus.

Field detection of Tritrichomonas foetus through loop mediated isothermal amplification with CELDA.

An affordable, portable, and easy-to-build instrument was implemented to perform loop-mediated isothermal amplification (LAMP) in the field. Controlled by Arduino, CELDA heats the reaction tubes, reads the intensity of green fluorescent light and stores data in a computer. In the laboratory and in the field, CELDA was successfully used for the detection of Tritrichomonas foetus DNA in less than 60 min and helped distinguish positives with less difficulty than visual inspection.

Taguchi method for the optimization of three loop mediated isothermal amplification procedures for Tritrichomonas foetus detection.

Taguchi method was used to optimize loop mediated isothermal amplification tests aimed to amplify segments of the elongation factor 1a1 (tf-ef1a1), the 5,8 ribosomal gene (tf-5,8 r) or the beta tubulin 2 (tf-btub2) from the protozoan parasite Tritrichomonas foetus. L9 orthogonal array and quadratic loss functions that penalize deviations from prediction values revealed the effect of amplification reaction components. Analysis of variance (ANOVA) decomposed the contribution of individual factors to a small Ct. Confirmation experiments established that optimum conditions were predictable, verifiable and reproducible. Primers concentration conditioned the non-specific amplification of tf-ef1a1 while betaine and magnesium concentration contributed to accelerate the time to reach a positive threshold in tf-ef1a1, tf-5,8 r and tf-btub2. The general strategy of simple and robust experimental design holds potential as a general optimization protocol for LAMP tests in every diagnostic laboratory.

Zero inflated count regression for one year prediction of bovine trichomonosis in a compulsory control plan in La Pampa, Argentine.

Data from 533 farms with bovine trichomonosis were investigated through hurdle and zero inflated models to quantify the burden of recurrent bovine trichomonosis. The probability of having a positive result in the following year for those farms with a previous positive test was 10.7%. Keeping or buying infected animals increased the odds of having positive results by 2.8 (95% CI = 1.41-5.56). The number first cases significantly decreased the chances of being no-at-risk (OR = 0.64; 95% CI = 0.47-0.89) and the chances of being positive in the following season (OR = 1.09; 95% CI = 1.01-1.18). The number of animals tested significantly increased the chances of being positive in the next season (OR = 1.02; 95% CI = 1.01-1.03). Both the number of positives and the number of animals tested suggest a significant proportion of new cases detected were false positives. These epidemiologic indicators are likely important determinants in the selection of farms requiring more intensive control measures and farms where testing results should be confirmed.

Major factors associated to persistence of bovine trichomoniasis in a mandatory control plan: A eight year retrospective study in La Pampa, Argentine.

Bovine trichomoniasis is a venereal disease caused by the flagellate protozoan Tritrichomonas foetus. Infection is related to low conception rates and would have significant impact on calf crop. The state of La Pampa started in 2006 an unprecedented mandatory control program for eradication of bovine trichomoniasis. The compulsory participation of all cattle producers and the yearly control of every bull should be followed by culling of every positive animal. This retrospective study on data from eight years of the control plan showed that 80% of farms had a single year of positive tests. In these farms, positive tests showed a strong decay of disease during the first years that reached a baseline by 2012. A non negligeable proportion of positive bulls in this group can be attributed to false positive tests. Oppositely, farms with two or more years of positive diagnosis accounted for a great proportion of recent cases. These farms were more likely related to less intensive control measures. The non exclusion of carrier bulls is the major factor contributing to the persistance of bovine trichomoniasis.

Direct detection of Tritrichomonas foetus in cattle genital fluid trough loop mediated isothermal amplification of elongation factor 1 alpha 1.

Testing for Tritrichomonas foetus and exclusion of infected animals is an effective way of improving the reproductive efficiency in a herd. Conventional PCR is inherently more specific than the culture method and quantitative PCR can significantly increase the detection limit. Loop Mediated Isothermal DNA Amplification (LAMP) is gaining interest because the method does not require expensive equipment, specificity and sensitivity can be as high as quantitative PCR. The object of this study was to develop a sensitive and friendly test for point-of-care detection of T. foetus. The LAMP test that targeted T. foetus elongation factor 1 alpha 1 sequences showed high specificity. Sensitivity was 100-1000 times higher than that reached through culture, polymerase chain reaction or with a previously developed LAMP for 5.8 ribosomal sequences. Moreover, T. foetus detection could be performed without DNA purification from infected cervical vaginal mucus (CVM) or smegma samples. The tf-ef1a1 LAMP method was tested for field detection with paper strips soaked in CVM from infected cows and the results were observed 90 min later. Direct detection of T. foetus in CVM with the tf-ef1a1 LAMP showed high sensitivity and specificity, and an overall diagnostic odds ratio of 56 (CI: 13.3-235.0). The tf-ef1a1 LAMP showed great potential for diagnosis and control of T. foetus in resource-challenged regions.

Evaluation of Tritrichomonas foetus infection clearance in heifers immunized with a single intravaginal dose of formaldehyde fixed strain B1 cells.

Vaccines against Tritrichomonas foetus have been shown to reduce the time of infection after natural or experimental exposure. The object of this study was to assess the protection against T. foetus infection conferred by a single vaginal instillation of formaldehyde fixed T. foetus cells. Aberdeen Angus virgin heifers were randomly allocated to 3 groups of 12 individuals to receive placebo or formaldehyde fixed T. foetus cells prepared following one of two procedures (formalin or freshly prepared solution) and six weeks later they were challenged with 106T. foetus trophozoites. The median time for clearance among control heifers was 93.75 days while in animals immunized with formaldehyde fixed T. foetus it was 45 days. A single vaginal dose of cells fixed with fresh formaldehyde solution gave a rate of decay of infection per unit of time of 2.54 (CI 95% = 1.07;6.01).

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).

Bacteria as source of diglycosidase activity: Actinoplanes missouriensis produces 6-O-α-L-rhamnosyl-ß-D-glucosidase active on flavonoids.

Bacteria represent an underexplored source of diglycosidases. Twenty-five bacterial strains from the genera Actinoplanes, Bacillus, Corynebacterium, Microbacterium, and Streptomyces were selected for their ability to grow in diglycosylated flavonoids-based media. The strains Actinoplanes missouriensis and Actinoplanes liguriae exhibited hesperidin deglycosylation activity (6-O-α-L-rhamnosyl-ß-D-glucosidase activity, EC 3.2.1.168), which was 3 to 4 orders of magnitude higher than the corresponding monoglycosidase activities. The diglycosidase production was confirmed in A. missouriensis by zymographic assays and NMR analysis of the released disaccharide, rutinose. The gene encoding the 6-O-α-L-rhamnosyl-ß-D-glucosidase was identified in the genome sequence of A. missouriensis 431(T) (GenBank accession number BAL86042.1) and functionally expressed in Escherichia coli. The recombinant protein hydrolyzed hesperidin and hesperidin methylchalcone, but not rutin, which indicates its specificity for 7-O-rutinosylated flavonoids. The protein was classified into the glycoside hydrolase family 55 (GH55) in contrast to the known eukaryotic diglycosidases, which belong to GH1 and GH5. These findings demonstrate that organisms other than plants and filamentous fungi can contribute to an expansion of the diglycosidase toolbox.

Evidence for repeated gene duplications in Tritrichomonas foetus supported by EST analysis and comparison with the Trichomonas vaginalis genome.

Tritrichomonas foetus causes a venereal infection in cattle; the disease has mild or no clinical manifestation in bulls, while cows may present vaginitis, placentitis, pyometra and abortion in the more severe cases. T. foetus has one of the largest known genomes among trichomonads. However molecular data are fragmentary and have minimally contributed to the understanding of the biology and pathogenesis of this protozoan. In a search of new T. foetus genes, a detailed exploration was performed using recently available expressed sequences. Genes involved in the central carbon metabolism (phosphoenol pyruvate carboxykinase, glyceraldehyde-3-phosphate dehydrogenase, fructose-1,6-bisphosphate aldolase, thioredoxin peroxidase, alpha and beta chains of succinyl CoA synthetase, malate dehydrogenase, malate oxidoreductase and enolase) as well as in cell structure and motility (actin, α-tubulin and ß-tubulin) were found duplicated and, in many cases, repeatedly duplicated. Homology analysis suggested that massive expansions might have occurred in the T. foetus genome in a similar way it was also predicted for Trichomonas vaginalis, while conservation assessment showed that duplications have been acquired after differentiation of the two species. Therefore, gene duplications might be common among these parasitic protozoans.

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.

Trombosis de varices orbitarias. Diagnóstico por tomografía computarizada / Thrombosis of orbital varices

Las várices orbitarias son malformaciones venosas producidas por una dilatación anormal de una o más venas orbitarias probablemente asociadas a debilidad congénita de la pared vascular. Son lesiones infrecuentes que se presentan en pacientes jóvenes y causan exoftalmos intermitente, relacionado a aumento en la presión venosa sistémica.Las complicaciones de las várices orbitarias son el sangrado y la trombosis y se manifiestan por exoftalmos de rápida evolución, dolor y disminución de la motilidad ocular.Presentamos dos casos de várices orbitarias complicadas por trombosis en pacientes adultos mayores, cuyo diagnóstico fue realizado por tomografía computarizada (TC). Se analizan los hallazgos ecográficos, de TC y resonancia magnética (RM) (AU)

Valor de la angiografía por tomografía computada en el diagnóstico de aneurismas intracraneanos / Intracraneal aneurysms: three-dimensional CT angiography

A fin de determinar el valor de la Angio TC en el diagnóstico de aneurismas intracraneanos se estudiaron 40 pacientes con cuadro clínico de HSA y/o imagen tomográfica compatible con aneurisma intracraneano. Se reconstruyeron las imágenes en el modo 3D de proyección de intensidad máxima (MIP). Treinta y siete de los pacientes fueron estudiados con angiografía por sustracción digital (ASD). En 34 de los 40 pacientes estudiados se realizó diagnóstico positivo de aneurisma intracraneano. Los 6 restantes presentaron Angio TC normal, siendo uno de ellos falso negativo, puesto en evidencia por la ASD. Concluimos que la Angio TC normal es un método rápido, poco invasivo y muy confiable para la detección de aneurismas intracraneanos que puede ser realizado a continuación del diagnóstico tomográfico de SAH, posibilitando así una cirugía de emergencia

Valor de la angiografía por tomografía computada en el diagnóstico de aneurismas intracraneanos / Intracraneal aneurysms: three-dimensional CT angiography

A fin de determinar el valor de la Angio TC en el diagnóstico de aneurismas intracraneanos se estudiaron 40 pacientes con cuadro clínico de HSA y/o imagen tomográfica compatible con aneurisma intracraneano. Se reconstruyeron las imágenes en el modo 3D de proyección de intensidad máxima (MIP). Treinta y siete de los pacientes fueron estudiados con angiografía por sustracción digital (ASD). En 34 de los 40 pacientes estudiados se realizó diagnóstico positivo de aneurisma intracraneano. Los 6 restantes presentaron Angio TC normal, siendo uno de ellos falso negativo, puesto en evidencia por la ASD. Concluimos que la Angio TC normal es un método rápido, poco invasivo y muy confiable para la detección de aneurismas intracraneanos que puede ser realizado a continuación del diagnóstico tomográfico de SAH, posibilitando así una cirugía de emergencia (AU)