Molecular Detection and Characterization of Ehrlichia canis Isolates from Three Geographic Regions in Mexico: A Retrospective Study.

Canine monocytic ehrlichiosis (CME) is the most common tick-borne disease affecting domestic dogs and other wild canids. It has a worldwide distribution and is associated with the presence of the brown dog tick. Few studies have been conducted in Mexico to identify and characterize Ehrlichia canis genetic variability. In the present study, 111 dogs of different sex, breed, and age from three geographic regions in Mexico were included. All of them had a previous history of tick infestation and/or the presence of one or more clinical signs compatible with CME. All dogs were tested by a commercial ELISA and nested PCR assay for the detection of E. canis. In addition, we analyzed the E. canis genetic diversity from the 16S rRNA gene sequences obtained in this study, along with 15 additional sequences described for E. canis in Mexico and obtained from GeneBank. Serological detection by commercial ELISA results showed overall infection rates of 85.58% (95/111), including 73.1% (30/41) in samples from Guerrero state; 75% (15/20) in Morelos; and 100% (50/50) in Chihuahua. On the other hand, molecular detection (nPCR assay) showed 31.5% (35/111) overall infection rate, with 41.4% (17/41) in Guerrero state; 55% (11/20) in Morelos; and 14% (7/50) in Chihuahua. We observed a high 16S rRNA gene sequence conservancy in most of the E. canis isolates in the three geographical areas from Mexico, including those analyzed in this research, suggesting a common geographic origin among isolates.

Molecular Identification of Babesia spp. and Anaplasma marginale in Water Buffaloes in Veracruz and Tabasco, Mexico: A Retrospective Study.

Two hundred and thirty-three blood samples of water buffalo were collected on four farms in Veracruz state and Tabasco state, Mexico, to detect and confirm the identities of Babesia and Anaplasma spp. sequences. Nested PCR assays were used for the amplification of specific genes encoding B. bovis rhoptry-associated protein (RAP-1), B. bigemina SpeI-AvaI restriction fragment, and Anaplasma marginale major surface protein 5 (MSP5). Using DNA sequencing and BLASTn analysis for DNA homology hemoparasite identification, the identities of the hemoparasites were established by comparing the nucleotide sequences obtained in this study with those available in the GenBank database at the National Center for Biotechnology Information (NCBI). Water buffalo infection with at least one of the hemoparasites under study was detected in 45% (105/233) of the blood samples, while a mixed infection with B. bovis and B. bigemina was detected in 6.4% (15/233) of samples. For this cross-sectional study, mixed infections with the three hemoparasites were not detected. BLASTn analysis revealed that the nucleotide sequences of the water buffalo isolates shared sequence identity values ranging from 88 to 100% with previously published gene sequences of B. bovis, B. bigemina, and A. marginale. The current results confirm that water buffalo, as cattle, are also carriers of hemoparasite infections that are tick-transmitted, and suggest that they probably have an important role in the epidemiology of bovine babesiosis in Mexico.

Possible Association between Selected Tick-Borne Pathogen Prevalence and Rhipicephalus sanguineus sensu lato Infestation in Dogs from Juarez City (Chihuahua), Northwest Mexico-US Border.

Tick-borne bacterial pathogens (TBBPs) show a worldwide distribution and represent a great impact on public health. The brown dog tick (Rhipicephalus sanguineus) is a vector of several pathogens that affect dogs and sometimes humans as well. In addition, TBBPs represent a diagnostic challenge and imply financial resources and medical treatment for long periods of time. In the present study, R. sanguineus s. l. was identified as the main tick species naturally parasitizing dogs that inhabit. Juárez City, Chihuahua, in the Paso del Norte region, Mexico-US Border, representing 99.8% of the cases. Additionally, an end-point PCR was performed to search for whether pathogens in R. sanguineus s. l. can transmit in DNA extracted from ticks and dog blood samples. This is the first molecular detection of Rickettsia rickettsi infecting domestic dogs in Mexico; however, other pathogens were also identified, such as Ehrlichia canis and Anaplasma platys in both ticks and dog blood samples, while Anaplasma phagocytophilum was identified only in dog blood samples. Moreover, co-detection in tick pools and co-infection in the analyzed dog blood samples could be found. Similarly, this research showed that dogs were found mostly parasitized by adult female ticks, increasing the possibility of transmission of E. canis.

Comparative Study of Indirect Fluorescent Antibody, ELISA, and Immunochromatography Tests for Serological Diagnosis of Bovine Babesiosis Caused by Babesia bovis.

The indirect fluorescent antibody test (IFAT) is the most frequently used test to conduct seroepidemiological studies so far, and it is regarded as the "gold standard" test for the serological diagnosis of bovine babesiosis. The aim of the present study was to compare the enzyme-linked immunosorbent assay (ELISA) and the rapid immunochromatography test (ICT) for use in the serological diagnosis of cattle exposed to B. bovis in Mexico. The evaluation of test performance was carried out with 30 positive and 30 negative reference sera. A total of 72 bovine sera samples collected from cattle in a region with endemic bovine babesiosis were analyzed by ELISA and ICT, and the results were compared with those of IFAT. Kappa value (k) was also calculated to determine the agreement between tests. The sensitivity and specificity of ELISA for detecting antibodies against B. bovis were 87% (26/30) and 80% (24/30), respectively. The sensitivity and specificity of ICT for detecting antibodies against B. bovis were 90% (27/30) and 83.3% (25/30), respectively. The overall concordance determined for ELISA and ICT was 94.4% (68/72) and 98.6% (71/72), respectively, when the results were compared with those of IFAT. ICT was more sensitive and specific in this comparative study, showing good strength of agreement (k = 0.79) with respect to IFAT. ICT combines a strip-based assay system that is fast, practical, and sensitive for detection of antibodies to B. bovis, which suggests that it could be applied in the field without requiring any laboratory equipment for its use and interpretation of test results.

Molecular Detection of Tick-Borne Pathogens in American Bison (Bison bison) at El Uno Ecological Reserve, Janos, Chihuahua, Mexico.

American bison (Bison bison) is listed as near-threatened and in danger of extinction in Mexico. Recent studies have demonstrated the presence of several emerging pathogens at the Janos Biosphere Reserve (JBR), inhabited by one wild herd of American bison. Blood samples were collected from 26 American bison in the JBR. We tested for the presence of Anaplasma marginale, Babesia bigemina, B. bovis, Borrelia burgdorferi sensu lato, and Rickettsia rickettsii DNA using nested and semi-nested PCR protocols performing duplicates in two different laboratories. Results showed three animals (11.5%) positive for B. burgdorferi s. l., three more (11.5%) for Rickettsia rickettsii, and four (19.2%) for B. bovis. Two individuals were co-infected with B. burgdorferi s. l. and B. bovis. We found no animals positive for A. marginale and B. bigemina. This is the first report in America of R. rickettsii in American bison. American bison has been described as an important reservoir for pathogens of zoonotic and veterinary importance; thus, the presence of tick-borne pathogen DNA in the JBR American bison indicates the importance of continuous wildlife health surveys.

Establishment of Babesia bovis In Vitro Culture Using Medium Free of Animal Products.

Babesia bovis, an etiological agent of bovine babesiosis, causes a significant burden to the cattle industry worldwide. The most efficient method to mitigate bovine babesiosis is a live vaccine produced by serial passage in splenectomized cattle. However, there are several concerns regarding live vaccine production, including variation between batches and the use of many animals. In this study, we report a B. bovis-SF strain continuously cultured in a medium free of components of animal origin enriched with a chemically defined lipid mixture (CD lipid mixture) and the use of a perfusion bioreactor to harvest a large amount of B. bovis. Six culture media were compared, including VP-SFM, CD-CHO, CD-Hydrolyzed, CD-CHO, SFM, and ADMEM/F12. We found that the VP-SFM medium performed the best for B. bovis growth, with a maximum percentage of parasitized erythrocytes (PPE) of 8.6%. The effect of six dilutions of a commercial mixture of CD lipids added to VP-SFM showed that the CD lipid mixture at a dilution of 1:100 had the best B. bovis growth curve, with a maximum PPE of 13.9%. Propagation of the in vitro B. bovis culture was scaled up in a perfusion bioreactor using VP-SFM with a CD lipid mixture, and the PPE reached over 32%. The continuous in vitro B. bovis culture in a medium free of animal origin components could potentially reduce and replace the use of animals to produce a reagent for diagnostics and live vaccines to control bovine babesiosis.

Demonstrating the presence of Ehrlichia canis DNA from different tissues of dogs with suspected subclinical ehrlichiosis.

BACKGROUND: Nowadays, Ehrlichia canis receives increasing attention because of its great morbidity and mortality in animals. Dogs in the subclinical and chronic phases can be asymptomatic, and serological tests show cross-reactivity and fail to differentiate between current and past infections. Moreover, there could be low parasitaemia, and E. canis might be found only in target organs, hence causing results to be negative by polymerase chain reaction (PCR) on blood samples. METHODS: We evaluated by PCR the prevalence of E. canis in blood, liver, spleen, lymph node and bone marrow samples of 59 recently euthanised dogs that had ticks but were clinically healthy. RESULTS: In total, 52.55% of the blood PCRs for E. canis were negative, yet 61.30% yielded positive results from tissue biopsies and were as follows: 63.15% from bone marrow; 52.63% from liver; 47.36% from spleen; and 15.78% from lymph node. In addition, 33% had infection in three tissues (spleen, liver and bone marrow). CONCLUSIONS: Our results show the prevalence of E. canis from tissues of dogs that were negative by blood PCR. Ehrlichia canis DNA in tissue was 30% lower in dogs that tested negative in PCR of blood samples compared to those that were positive. However, it must be taken into account that some dogs with negative results were positive for E. canis in other tissues.

Innovative Alternatives for Continuous In Vitro Culture of Babesia bigemina in Medium Free of Components of Animal Origin.

In this study, we report Babesia bigemina proliferation in culture medium free of components of animal origin supplemented with a lipid mixture. Babesia bigemina continuously proliferated in VP-SFM with a higher percent parasitized erythrocyte as compare to using other animal component-free culture media. Compared with Advanced DMEM/F12 (ADMEM/F12), VP-SFM had a similar percent parasitized erythrocyte (PPE). Supplementation of VP-SF with a lipid acid mixture improved B. bigemina proliferation in vitro culture, with a maximum PPE of 11.3%. Growth of B. bigemina in a perfusion bioreactor using VP-SFM medium supplemented with lipid mixture resulted in a PPE above 28%. In conclusion, we demonstrated that B. bigemina proliferated in an animal component-free medium supplemented with the fatty acid mixture. This innovation to B. bigemina in vitro culture method presented herein is an important source of biological material for live vaccine production and understanding the mechanisms and molecules involved in parasite attachment and invasion of bovine erythrocytes.

La vacunación simultánea de bovinos con Lactobacillus casei y la vacuna bivalente contra babesiosis bovina genera una mejor protección contra Babesia bovis y B. bigemina transmitidas por garrapatas en condiciones extremas de campo / The simultaneous vaccination of bovines with Lactobacillus casei and the bivalent vaccine against bovine babesiosis induces a better protection against Babesia bovis and B. bigemina transmitted by ticks in extreme field conditions

The effect of Lactobacillus casei on INIFAP's mixed vaccine against bovine babesiosis (VAC) was assessed in bovines in an endemic babesiosis area. It was previously reported that L. casei increases the efficiency of the Mexican mixed vaccine against bovine babesiosis under controlled conditions. The results of the present study demonstrated the effectiveness of simultaneous vaccination of bovines with L. casei and the mixed vaccine against bovine babesiosis in eliciting a protective immune response under extreme conditions in the field. Twenty Babesia spp free bovines were allocated into three groups: un-vaccinated (Control, n = 9), vaccinated with VAC (n = 5), and vaccinated simultaneously with VAC and Lactobacillus casei (LC-VAC, n = 6). All animals were kept in a tick and Babesia spp free field at Coatepec, Veracruz during 24 days before moving them to Paso del Toro, Veracruz, for a natural exposition to Babesia spp transmitted by Riphicephalus (Boophilus) microplus ticks. Protection against Babesia spp was observed in bovines belonging to VAC and LC-VAC groups, while control animals showed severe clinical babesiosis. Bovines in VAC-LC group showed less clinical signs between days 12-16 after challenge as compared with animals in VAC group. All bovines showed both Babesia spp after challenge. Levels of IgG anti-Babesia in animals from both vaccinated groups, determined by indirect immunofluorescence test, always were higher to Babesia bovis than to B. bigemina after vaccination and challenge. It was demonstrated the efficiency of simultaneous vaccination of bovines with VAC and L. casei, in eliciting a better protective immune response against naturally transmitted Babesia spp under extreme field conditions.

Se evaluó el efecto de Lactobacillus casei en la vacuna mixta contra babesiosis bovina del INIFAP (VAC), en bovinos de un área endémica de babesiosis. Previamente se informó que L. casei incrementa la eficacia de la vacuna mixta mexicana contra babesiosis bovina bajo condiciones controladas. Los resultados aquí expuestos demostraron dicha efectividad para generar una respuesta inmunitaria protectora bajo condiciones extremas en el campo. Veinte bovinos libres de Babesia spp fueron distribuidos al azar en tres grupos: testigo no vacunado (Testigo, n = 9), vacunado con VAC (n = 5), y vacunado simultáneamente con VAC y L. casei (LC-VAC, n = 6). Todos los animales se mantuvieron en un corral libre de garrapatas y Babesia spp en Coatepec, Veracruz durante 24 días antes de transportarlos a Paso del Toro, Veracruz, para una exposición natural a Babesia spp transmitida por garrapatas Riphicephalus (Boophilus). Se observó protección contra Babesia spp en bovinos pertenecientes a los grupos VAC y LC-VAC, mientras que los animales testigo mostraron signos clínicos de babesiosis aguda. Los bovinos del grupo VAC-LC mostraron menos signos clínicos que los del grupo VAC entre los días 12-16. Todos los bovinos mostraron Babesia spp después de la confrontación. Los niveles de IgG anti-Babesia en los animales de los grupos vacunados, determinados por inmunofluorescencia indirecta, siempre fueron más elevados contra Babesia bovis que contra B. bigemina después de la vacunación y de la confrontación. Se demostró la eficacia de la vacunación simultánea con VAC y L. casei en bovinos, para generar una mejor respuesta inmunitaria protectora contra Babesia spp transmitida naturalmente por garrapatas, bajo condiciones extremas de campo.

Nilgai antelope in northern Mexico as a possible carrier for cattle fever ticks and Babesia bovis and Babesia bigemina.

Of 20 blood samples from nilgais from México, five were polymerase chain reaction-positive for Babesia bigemina and one for Babesia bovis. Positive samples had the expected 170 (B. bigemina) and 291 (B. bovis) base pairs and were identical to Gen-Bank B. bigemina accession S45366 and B. bovis M38218.