Microfilaremic infection in canine filariosis in Colombia: a challenge in morphological and molecular diagnostics.

Canine filariosis is caused by filiform nematodes and affects several species of animals as well as humans. The disease produces a wide range of symptoms that can often be confused with other diseases, which increases the complexity of its diagnosis. The search for methodologies to facilitate its diagnosis is a challenge, and specific and differential identification of the parasite species causing the disease holds key to a successful diagnosis. In Colombia, there is a problem of underdiagnosis of filariosis in microfilaremic dogs infected by Dirofilaria immitis and Acanthocheilonema reconditum, and of microfilaremias not related to heartworm disease. The highest prevalences have been reported for D. immitis infections, although new cases of A. reconditum infections are beginning to appear. The aim of this study was to differentiate the microfilariae infections caused by D. immitis and A. reconditum by a morphological and molecular characterization of microfilariae so as to facilitate an accurate diagnosis of canine filariosis in the metropolitan area of Bucaramanga (Colombia). For this purpose, 400 blood samples with anticoagulants were collected from the dogs and analyzed with the help of a commercial immunochromatography kit for the detection of D. immitis circulating antigen. The Woo, Knott, and polymerase chain reaction (PCR) techniques were employed for determining the parasite count, morphological observation, and molecular identification of microfilariae present in the dogs respectively. The prevalence of microfilaremic dogs in Bucaramanga metropolitan area was 18.75% (75/400). The prevalence of dogs that tested positive for D. immitis in the antigen and in PCR tests was 1.25% (5/400) and 1% (4/400), respectively. Furthermore, the PCR test revealed that 17.75% of the microfilaremic dogs tested positive for A. reconditum (71/400) (first report in the metropolitan area of Bucaramanga), with one animal co-infected by both species, and 0% for D. repens (0/400). However, by morphological characterization, 4% of the microfilariae (3/75) corresponded to D. immitis, 20% (15/75) to D. repens, and 76% (57/75) to A. reconditum. The use of molecular diagnostic methods such as PCR aids in the specific identification of the parasite, thus making it a more accurate method than the morphological characterization of microfilariae. The identification of the parasites by PCR helps improve the veterinary diagnosis of canine filariosis in Colombia, which would lead to the establishment of an appropriate treatment protocol for each species of filaria and also to the generation of reliable data to be used at the clinical and epidemiological levels.

Epidemiological assessment of Anaplasma marginale, Babesia bigemina, and Babesia bovis infections in Colombian creole cattle breeds: A molecular survey in northeastern Colombia.

Anaplasmosis and babesiosis are globally distributed arthropod-borne diseases known for causing substantial economic losses due to their high morbidity and mortality rates. This study aims to assess the frequency and epidemiological features associated with the infection of Anaplasma marginale, Babesia bigemina, and Babesia bovis in three Creole cattle breeds (Chino Santandereano (Chino), Casanareño (CAS), and Sanmartinero (SM)) in northeastern Colombia. Between June 2019 and March 2020, a total of 252 Creole cattle were sampled, with Chino, CAS, and SM accounting for 42.8%, 29.5%, and 29.5% of the samples, respectively. Blood samples were subjected to molecular analysis to detect the DNA of A. marginale, B. bigemina, and B. bovis, using species-specific primers. Additionally, Packed Cell Volume (PCV), total serum proteins, and body condition were evaluated. Molecular analyses revealed the presence of B. bigemina, A. marginale, and B. bovis in 83.7% (211/252; 95% CI = 79.1%-88.3%), 59.9% (151/252; 95% CI = 53.8%-66.1%), and 40.9% (103/252; 95% CI = 34.7%-46.9%) of the samples, respectively, with 69% (174/252; 95% CI = 57.8%-80.3%) exhibiting coinfections. Notably, in infected animals, no significant alterations in PCV, total serum proteins, or body condition were observed. Multivariate analyses indicated a statistically significant association between the frequency of A. marginale infection and the breed and season, with a higher frequency in SM during the rainy season (P < 0.05). To our knowledge, this is the first molecular survey that evaluates multiple arthropod-borne pathogens in Colombian Creole breeds. The results revel a high frequency of B. bigemina and A. marginale infections, coupled with a notable frequency of coinfections, all without significant alteration in the PCV, total serum proteins and body conditions. Our findings enhance the understanding of the epidemiological aspects of arthropod-borne pathogens in Colombian Creole breed and contribute to the improvement of sanitary programs for these animals.

Enhancing Trypanosomatid Identification and Genotyping with Oxford Nanopore Sequencing: Development and Validation of an 18S rRNA Amplicon-Based Method.

Trypanosomatids, including Trypanosoma and Leishmania species, present significant medical and veterinary challenges, causing substantial economic losses, health complications, and even fatalities. Diagnosing and genotyping these species and their genotypes is often complex, involving multiple steps. This study aimed to develop an amplicon-based sequencing (ABS) method using Oxford Nanopore long-read sequencing to enhance Trypanosomatid detection and genotyping. The 18S rDNA gene was targeted for its inter-species conservation. The Trypanosomatid-ABS method effectively distinguished between 11 Trypanosoma species (including Trypanosoma evansi, Trypanosoma theileri, Trypanosoma vivax, and Trypanosoma rangeli) and 6 Trypanosoma cruzi discrete typing units (TcI to TcVI and TcBat), showing strong concordance with conventional methods (κ index of 0.729, P < 0.001). It detected co-infections between Trypanosomatid genera and T. cruzi, with a limit of detection of one parasite per mL. The method was successfully applied to human, animal, and triatomine samples. Notably, TcI predominated in chronic Chagas samples, whereas TcII and TcIV were found in the acute stage. Triatomine vectors exhibited diverse Trypanosomatid infections, with Triatoma dimidiata mainly infected with TcI and occasional TcBat co-infections, and Rhodnius prolixus showing TcI and TcII infections, along with T. rangeli co-infections and mixed TcII infections. Animals were infected with T. vivax, T. theileri, and T. evansi. The ABS method's high resolution, sensitivity, and accuracy make it a valuable tool for understanding Trypanosomatid dynamics, enhancing disease control strategies, and enabling targeted interventions.

An Outbreak of Acute Chagas Disease Possibly Spread through Oral Transmission Involving Animal Reservoirs in Eastern Colombia.

Chagas disease (CD) is a parasitic infection caused by the parasite Trypanosoma cruzi. Reports of CD cases associated with oral transmission have increased, particularly in Colombia, Brazil, and Venezuela. In this investigation, parasitological, serological, and molecular tests were conducted on samples obtained from humans, mammal reservoirs, and hosts involved in the assessment of a suspected oral transmission outbreak in Cubara, Boyaca, Colombia. Seropositivity was observed in 60% (3 of 5) of index patients and 6.4% (5 of 78) of close contacts. Trypanosoma cruzi DNA was detected by quantitative polymerase chain reaction in 100% of index cases, 6.4% (5 of 78) of close contacts, 60% (6 of 10) of canines, and 100% (5 of 5) of opossums. In all index cases, the TcI lineage was identified, along with two cases of mixed infection (TcI/TcII-TcVI). Hemoculture revealed a flagellate presence in 80% of opossums, whereas all triatomine bugs tested negative. Our findings suggest a potential oral transmission route through contamination with opossum secretions.

Epidemiological features of Leishmania infantum in dogs (Canis lupus familiaris) suggest a latent risk of visceral leishmaniasis in the metropolitan area of Bucaramanga, Santander, Eastern Colombia.

Visceral leishmaniasis (VL) is a disease caused by species of the Leishmania donovani complex that is mainly transmitted through the urban cycle involving dogs as the primary reservoir. In Colombia, the incidence of VL is increasing, along with the spread of potential vectors. This study aims to investigate the eco-epidemiological factors associated with Leishmania spp. infection in dogs from the Metropolitan Area of Bucaramanga (MAB), Santander, eastern Colombia, which is a region at risk for VL. We conducted molecular and serological surveillance of Leishmania spp. in 207 dogs from MAB to determine the epidemiological factors associated with infection. Subsequently, we carried out a molecular and serological analysis of phlebotomine and humans, respectively, in areas with a higher prevalence of infection, aiming to describe the main features associated with the transmission cycle. Out of the 207 dogs tested, 37 (17.8%, 95% CI = 12.6-23.1%) were positive for the presence of Leishmania antibodies by the IFAT test, and only 9 (4.3%, 95% CI = 1.55-7.15%) were positive for L. infantum by PCR. Multivariate analyses indicated that canine shelters and dogs with clinical signs commonly associated with canine VL had a higher prevalence of infection (P < 0.05). In the entomological survey, 69 blood-fed female phlebotomine of the genus Lutzomyia were captured in canine shelters, among them, 55% were identified as Lutzomyia camposi, 29% as Lu. ovallesi, 7% as Lu. dubitans, 6% as Lu. torvida, and 3% as Lu. cayennensis. The identified meal sources of the phlebotomine included human, pig, avian, cattle, and porcupine (Coendou quichua) blood. However, no phlebotomine positive for Leishmania spp. were detected by molecular analyses. Finally, 14 humans who had frequent contact with L. infantum-positive dogs were analyzed through rK39 test, but none tested was positive for IgG/IgM antibodies. The molecular and serological analyses indicate the circulation of L. infantum in dogs from MAB, with canine shelters having the highest prevalence of infection. The entomological survey of canine shelters showed a significant diversity of phlebotomine without potential vectors of L. infantum, suggesting the presence of infection in dogs from these areas could take place in other locations or through other transmission routes. The circulation of L. infantum in multiple dogs from MAB suggests a latent risk of zoonotic transmission of VL in these cities.

Clinical and epidemiological aspects of the infection by Babesia, Theileria and Trypanosoma species in horses from northeastern Colombia.

Piroplasmosis and trypanosomiasis are debilitating diseases of great economic impact on the equine industry of Latin America. Considering the lack of studies in the northeastern part of Colombia, this study aimed to determine the epidemiological, clinical and genetic features associated with infection of the Babesia, Theileria, and Trypanosoma species in horses from this geographical area. Two hundred and eighty horses from the Arauca, Meta, and Santander departments were molecularly analyzed for infection with Babesia caballi, Theileria equi, Trypanosoma evansi, and Trypanosoma vivax. Furthermore, clinical, epidemiological and entomological analyses were performed on the data sets. Molecular analysis showed 25.7% and 3.9% prevalence for T. equi and T. evansi, respectively, without positive animals for B. caballi and T. vivax. There were no differences in the prevalence of T. equi between departments, whereas T. evansi was detected exclusively in Santander. A total of 633 ticks were collected from 72 horses across the three departments, with 84.7% corresponding to Dermacentor nitens, 10.9% to Amblyomma cajennense (sensu lato) (s.l). and 4.4% to Rhipicephalus microplus. For T. equi, genetic analyses showed that Colombian isolates belong to genotype C of species, along with sequences of Brazil and Mexico. Epidemiological analysis revealed a significant association between tick infestation and lack of vector control with molecular infection of T. equi, whereas clinical analysis revealed a significant reduction in packed cell volume, red blood cells, and mean corpuscular volume in positive animals to this pathogen. Furthermore, molecular infection by T. evansi was associated with epidemiological characteristics in the Santander department. In conclusion, our analysis revealed a moderate infection rate by T. equi of genotype C in horses from northeastern Colombia, which affects their clinical conditions. Control of ticks and treatment of symptomatic animals should be considered to reduce the economic impact associated with these infections in the equine industry.

Molecular characterization of Ehrlichia canis and Babesia vogeli reveals multiple genogroups associated with clinical traits in dogs from urban areas of Colombia.

Ehrlichia canis and Babesia vogeli are vector-borne pathogens that infect blood cells and produce the diseases Canine Monocytic Ehrlichiosis (CME) and Babesiosis in dogs. Considering the lack of studies on these pathogens in Colombia, this study aims to determine the molecular prevalence and genetic characterization of E. canis and Babesia spp., in dogs from the Metropolitan Area of Bucaramanga (MAB), Santander, a region with one of the greatest pet densities in Colombia. One hundred eighty-five dogs were surveyed and analyzed through molecular, clinical, and hematological approaches. The molecular detection of E. canis and Babesia spp., was performed by conventional PCR targeting the dsb and 18S rRNA genes, respectively. To identify genogroups, E. canis positive samples underwent a hemi-nested PCR of the trp36 gene, and the PCR products were subsequently sequenced. Molecular analyses showed a prevalence of 13% (24/185; CI 95%, 8.1 - 18.0%) and 1.09% (2/185; CI 95,% -0.43 - 2.6%) for E. canis and B. vogeli respectively, as well as the presence of the genogroups US (USA), BR (Brazil), and CR (Costa Rica), in 62.5, 16.6, and 16.6% of E. canis positive samples, respectively. Values of hematocrit, hemoglobin, platelets, erythrocytes, white blood cell (WBC) count, lymphocytes, and eosinophils showed significant differences between animals infected with the different genogroups of E. canis (p< 0.05). In contrast, hematocrit values, hemoglobin, platelets, red blood cells, and creatine kinase MB isoenzyme (CK-MB) were lower in B. vogeli positive animals. Statistical analysis indicated that E. canis infection was associated with specific socioeconomic sectors as well as with some household features (p< 0.05). In conclusion, our results present evidence of the circulation of multiple genogroups of E. canis in the MAB, which is associated with different geographical origins and clinical traits. Epidemiological analyses suggest a need to increase molecular surveillance and prevention campaigns especially in lower socioeconomic sectors.

An overview of the trypanosomatid (Kinetoplastida: Trypanosomatidae) parasites infecting several mammal species in Colombia.

BACKGROUND: Trypanosomatids are among the most critical parasites for public health due to their impact on human, animal, and plant health. Diseases associated with these pathogens manifest mainly in poor and vulnerable populations, where social, environmental, and biological factors modulate the case incidence and geographical distribution. METHODS: We used Sanger and amplicon-based next-generation sequencing (NGS) in samples from different mammals to identify trypanosomatid infections in several departments in Colombia. A total of 174 DNA samples (18 humans, 83 dogs, and 73 wild mammals) were analyzed by conventional PCR using a fragment of the heat shock protein 70 (Hsp70) gene and Sanger sequenced the positive samples. Twenty-seven samples were sent for amplicon-based NGS using the same gene fragment. Data obtained were used to perform diversity analyses. RESULTS: One hundred and thirteen samples were positive for PCR by Hsp70 fragment; these corresponded to 22.1% Leishmania spp., 18.6% L. amazonensis, 9.7% L. braziliensis, 14.2% L. infantum, 8% L. panamensis, and 27.4% Trypanosoma cruzi. Comparison of the identified species by the two sequencing technologies used resulted in 97% concordance. Alpha and beta diversity indices were significant, mainly for dogs; there was an interesting index of coinfection events in the analyzed samples: different Leishmania species and the simultaneous presence of T. cruzi and even T. rangeli in one of the samples analyzed. Moreover, a low presence of L. braziliensis was observed in samples from wild mammals. Interestingly, to our knowledge, this is the first report of Leishmania detection in Hydrochaeris hydrochaeris (capybara) in Colombia. CONCLUSIONS: The Hsp70 fragment used in this study is an optimal molecular marker for trypanosomatid identification in many hosts and allows the identification of different species in the same sample when amplicon-based sequencing is used. However, the use of this fragment for molecular diagnosis through conventional PCR should be carefully interpreted because of this same capacity to identify several parasites. This point is of pivotal importance in highly endemic countries across South America because of the co-circulation of different genera from the Trypanosomatidae family. The findings show an interesting starting point for One Health approaches in which coevolution and vector-host interactions can be studied.

Human urogenital myiasis caused by the 'rat-tailed' larvae of Palpada scutellaris (Fabricius, 1805) in Santander, eastern Colombia: A case report.

The Palpada genus, which belongs to the Diptera order (family, Syrphidae), has been rarely reported to cause accidental myiasis in humans. Herein, we report the first case of genitourinary myiasis caused by a larva of the Palpada genus in a 9-year-old girl from Colombia. The girl, who resided in a rural area in the municipality of Floridablanca, Santander, near Bucaramanga city, in eastern Colombia, presented with lower abdominal pain accompanied by oliguria, followed by the subsequent elimination of a larva through the urine. The next day, the patient visited a primary healthcare centre, and no signs or symptoms were observed on clinical examination. Haematological analysis showed high plateletcrit levels and platelet large cell counts. The results of the urine test revealed a decrease in specific gravity and a slight increase in bacterial content and mucus. DNA barcoding analyses showed that the etiological agent corresponded to a third instar larva of the Palpada scutellaris species. This is the first case to report genitourinary myiasis caused by larvae of the genus Palpada in humans. However, we believe that additional cases might be accurately detected if adequate tests are performed to confirm the clinical and molecular features associated with this infection.

Distribution and natural infection status of synantrophic triatomines (Hemiptera: Reduviidae), vectors of Trypanosoma cruzi, reveals new epidemiological scenarios for chagas disease in the Highlands of Colombia.

INTRODUCTION: Updating the distribution and natural infection status of triatomine bugs is critical for planning, prioritizing, and implementing strategies to control Chagas disease (CD), especially after vector reduction programs. After carrying out a control program, the Department of Boyaca contains the highest number of Colombian municipalities certified by PAHO to be free of intradomiciliary transmission of Trypanosoma cruzi by Rhodnius prolixus. The present work describes the spatial distribution, natural infection (NI), and molecular characterization of T. cruzi in synanthropic triatomines from the Department of Boyaca in 2017 and 2018. MATERIALS AND METHODS: An entomological survey was conducted in 52 municipalities in Boyaca known to have had previous infestations of triatomine bugs. Insects were collected through active searches carried out by technical personnel from the Secretary of Health and community members using Triatomine Collection Stations (PITs-acronym in Spanish). For evaluation of natural infection, triatomines were identified morphologically and grouped in pools of one to five individuals of the same species collected in the same household. DNA derived from the feces of each pool of insects was analyzed by PCR for the presence of T. cruzi using primers flanking the satellite DNA of the parasite. SL-IR primers were used to differentiate TCI from the other DTUs and to identify different genotypes. The distribution of the collected triatomines was analyzed to determine any vector hotspots using spatial recreation. RESULTS: A total of 670 triatomine bugs was collected, belonging to five species: Triatoma dimidiata (73.2%), Triatoma venosa (16.7%), Panstrongylus geniculatus (5.7%), Rhodnius prolixus (4.4%), and Panstrongylus rufotuberculatus (0.4%), from 29 of the 52 municipalities. In total, 71.6% of the bugs were collected within houses (intradomiciliary) and the rest around the houses (peridomiciliary). Triatoma dimidiata was the most widely distributed species and had the highest natural infection index (37.8%), followed by T. venosa and P. geniculatus. TcI was the only DTU found, with the TcI Dom genotype identified in 80% of positive samples and TcI sylvatic in the other insects. Spatial analysis showed clusters of T. dimidiata and T. venosa in the northeast and southwest regions of Boyaca. CONCLUSIONS: After some municipalities were certified free of natural transmission within houses (intradomiciliary transmission) of T. cruzi by R. prolixus, T. dimidiata has become the most prevalent vector present, and represents a significant risk of resurgent CD transmission. However, T. venosa, P. geniculatus, and P. rufotuberculatus also contribute to the increased risk of transmission. The presence of residual R. prolixus may undo the successes achieved through vector elimination programs. The molecular and spatial analysis used here allows us to identify areas with an ongoing threat of parasite transmission and improve entomological surveillance strategies.

Molecular surveillance of Trypanosoma spp. reveals different clinical and epidemiological characteristics associated with the infection in three creole cattle breeds from Colombia.

In South America, Colombia is the third-largest livestock producer with approximately 28.8 million cattle, of which Colombian Creole cattle represent around 1% of the livestock population. Animal Trypanosomiasis (AT) is one of the most critical problems in the livestock industry, reducing its production by about 30 %. Considering the paucity of information to understand the epidemiological features of AT in Colombian Creole cattle, the present study reports the molecular prevalence and clinical traits associated with the infection of Trypanosoma spp. in three Colombian Creole breeds. From 2019 to 2020, cross-sectional surveillance in farms of central and west of Colombia was designed to evaluate the mentioned characteristics in Casanareño, Chino Santandereano, and Sanmartinero Creole breeds. Molecular analysis showed an AT prevalence of 60.2 % (95 % CI = 54.2 % - 66.2 %). The Chino Santandereano population presented the highest value (Trypanosoma spp., 75.2 %, T. theileri 59.6 % and T. evansi 15.6 %), followed by Casanareño (Trypanosoma spp., 65.3 %, T. theileri 38.6 %, T. evansi 24.0 %, and T. vivax 5.3 %) and Sanmartinero (Trypanosoma spp., 33.3 %, T. theileri 24.0 % and T. evansi 9.3 %). Features such as breeds, age, and feeding system were significantly associated with AT prevalence (P < 0.05). Additionally, a low level of serum total proteins was observed during T. evansi infection in Sanmartinero (P < 0.05). To our knowledge, this is the first cross-sectional survey that evaluates using molecular methods the infection of Trypanosoma spp. in Colombian Creole breeds, showing significant variations in the prevalence and clinical signs associated with the infection. These results suggest different degrees of trypanotolerance in these breeds, as well as a possible effect of environmental variables on the prevalence and clinical characteristics associated with the infection. The epidemiological and economic implications of these findings are discussed here.

Molecular surveillance reveals bats from eastern Colombia infected with Trypanosoma theileri and Trypanosoma wauwau-like parasites.

Several species of trypanosomes can infect bats (Chiroptera), but current information about bat trypanosomes in Colombia is scarce. The objectives of this study were to estimate the infection rate and to characterize the trypanosome species infecting bats from three rural regions near the municipality of Cumaribo in Vichada, Colombia. Blood samples were collected from 39 bats. DNA was extracted from the blood samples and analyzed using nuclear genetic markers (SSU rDNA, ITS rDNA, and cathepsin genes) to discriminate among trypanosome species. Trypanosomes were detected in 66.7 % (26/39) of blood samples using PCR; 61.5 % (24/39) of infections were identified as Trypanosoma theileri and 5.1 % (2/39) as T. wauwau-like parasites. The phylogeographic analysis revealed that our T. theileri sequences were associated with the TthIIB genotype from cattle in Brazil and Venezuela. The T. wauwau-like parasites represent a new genotype of the species and were found in Molossus molossus and Platyrrhinus helleri bats. These data represent the first evidence of this trypanosome in both Colombia, and in these species of bats. Bat infections with T. theileri suggest an important role of these hosts in maintaining this genotype, probably acquired by ingesting insect vectors. The T. wauwau-like genotype in new mammalian host species supports the 'bat seeding' hypothesis of the T. cruzi clade. The epidemiological and evolutionary implications of these findings are discussed.

Epidemiological and clinical characteristics of Trypanosoma cruzi infection in dogs (Canis lupus familiaris) from a Chagas Disease-Endemic Urban Area in Colombia.

In the last few years, an unusual increase in the number of acute Chagas disease outbreaks, presumably due to oral transmission, has been reported in urban areas in Santander, Colombia. Given the importance of dogs (Canis lupus familiaris) as reservoir hosts and sentinels of T. cruzi infection across different regions of America, we carried out a serological and molecular survey on T. cruzi infection in 215 dogs from the metropolitan area of Bucaramanga, Santander. Serological detection was carried out using enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence antibody test (IFAT), and indirect hemagglutination assay (IHA), while molecular detection was done using a nested PCR (nPCR), targeting the microsatellite region of T. cruzi nuclear DNA. Animals were defined as seropositive when at least two of the three serological tests were positive, and only these animals were evaluated with the nPCR. To discriminate DTU TcI from other DTUs, a multiplex PCR was performed in the T. cruzi-positive samples. Additionally, clinical and hematological traits were evaluated in these hosts. The dog sera showed a seropositivity rate of 27.9 % (60/215), of which 43.3 % (26/60) were positive for nPCR. Statistical analysis indicated that T. cruzi seropositive in dogs was associated with specific socioeconomic sectors and a lack of garbage collection in these municipalities. Hematological analyses showed that T. cruzi infection was associated with anemia and platelet alterations but not with alterations of aspartate aminotransferase (ASAT) and creatine kinase myocardial band (CK-MB). The high seroprevalence of infection and active circulation of T. cruzi I (TcI) in dogs reflect the risk of infection to humans in this area, which should be taken into consideration when Chagas disease control programs are implemented. In addition, T. cruzi infection may take a toll on dog health, which should be considered during dog care and management.

Caracterización de decomisos de vísceras rojas en un frigorífico de exportación en Santander - Colombia / Characterising red viscera confiscation in an export slaughterhouse in Santander, Colombia / Caracterização de confiscos de vísceras vermelhas num frigorífico de exportação em Santander - Colômbia

Resumen A nivel mundial el decomiso de vísceras rojas es una de las principales causas de pérdidas económicas en la comercialización de productos cárnicos. El objetivo del presente estudio fue determinar las principales causas y variables epidemiológicas asociadas al decomiso de vísceras rojas en el FRIGORÍFICO RÍO FRÍO (FRF), del municipio de Floridablanca, Santander y estimar el valor de las pérdidas económicas asociadas a esta actividad. Entre 9 de noviembre al 9 de diciembre de 2018, se realizó un estudio transversal en el total de animales sacrificados en el FRF, a los cuales se les recolectó información epidemiológica posterior a la inspección sanitaria realizada por funcionarios del INVIMA. Las principales causas y variables epidemiológicas asociadas al decomiso de vísceras rojas se estimaron a partir de análisis de frecuencias, mientras el valor de las pérdidas económicas, a través de un análisis económico en el mercado nacional. Se sacrificaron un total de 8671 bovinos, y 226 búfalos, en los cuales se realizó el decomiso de vísceras rojas en el 15,8% y 26,5% de los animales, respectivamente. En los bovinos los principales órganos decomisados fueron pulmón, hígado y bazo con porcentajes del 87,4%, 11,1% y 0,6%, respectivamente. Mientras que, en los búfalos, fueron el pulmón, hígado y riñón con porcentajes del 88,3%, 8,3% y 1,7%, respectivamente. Las principales causas de decomiso de pulmón en ambas especies fueron la broncoaspiración (86,6%) y enfisema (9,4%), mientras para el hígado fueron los abscesos (40,5%) y la distomatosis hepática (28,8%) en los bovinos, hígado graso (80,0%) y cirrosis (20,0%) en los búfalos. Para el caso de los bovinos, hubo diferencias significativas en la proporción de animales con distomatosis hepática entre los diferentes municipios de Santander (χ² = 50.03, P < 0.05). Las mayores pérdidas económicas durante el periodo de estudio se deben al decomiso de pulmón e hígado con valores de 4886 y 2639 USD. En conclusión, el pulmón fue el órgano decomisado con mayor frecuencia en ambas especies, principalmente asociado a la broncoaspiración. Futuras estrategias de intervención, con el fin de reducir la broncoaspiración en los animales que ingresan a la planta de beneficio, e interrumpir el ciclo de vida de Fasciola hepatica en algunos municipios de Santander, son necesarios para disminuir el nivel de pérdidas económicas a lo largo de la cadena productiva.

Abstract Red viscera confiscation represents a significant cause of economic loss for the meat products' trade. This study was aimed at determining the causes and epidemiological variables associated with red viscera confiscation in a slaughterhouse (FRIGORÍFICO RÍO FRÍO (FRF)) located in Floridablanca, in Colombia's Santander department, and estimating the value of the economic losses associated with such confiscation. A cross-sectional study was carried out between November and December 2018 based on epidemiological information regarding all animals slaughtered at the FRF recorded after routine health inspection by Colombian Food and Drug Surveillance Institute (INVIMA) officials. Frequency analysis was used for estimating the main causes of and the epidemiological variables associated with red viscera confiscation whilst economic losses were estimated by national market analysis. It was found that red viscera were confiscated in 15.8% of 8,671 sacrificed cattle compared to 26.5% for 226 buffaloes. The lungs (87.4%), liver (11.1%) and spleen (0.6%) were the main organs seized for cattle and lungs (88.3%), liver (8.3%) and kidneys (1.7%) for buffaloes. Bronchoaspiration (86.6%) and emphysema (9.4%) were the main causes of confiscation regarding the lungs in both species while for the liver these were abscesses (40.5%) and hepatic dystomatosis (28.8%) in cattle and fatty liver (80.0%) and cirrhosis (20.0%) in buffaloes. There were significant differences regarding the percentage of cattle having hepatic dystomatosis amongst municipalities in the Santander department (χ²=50.03, p<0.05). Lung (4,886 USD) and liver confiscation (2,639 USD) led to the greatest economic losses; the lungs were the main organ confiscated in both species, mainly being associated with bronchoaspiration. Future intervention strategies are needed to reduce the livestock industry's financial losses, aimed at reducing bronchoaspiration in animals in the slaughterhouse and interrupting Fasciola hepatica's life-cycle in some municipalities in the Santander department.

Resumo Globalmente, os confiscos de órgãos e vísceras vermelhas é uma das principais causas de perdas econômicas na comercialização de produtos à base de carne. O objetivo deste estudo foi determinar as principais causas e variáveis epidemiológicas associadas aos confiscos de vísceras vermelhas num frigorífico (FRIGORÍFICO RÍO FRÍO (FRF)), do município de Floridablanca, Santander, e estimar o valor das perdas econômicas associadas a esta atividade. Entre 9 de novembro e 9 de dezembro do 2018, foi realizado um estudo transversal sobre o total de animais abatidos no FRF, nos quais a informação epidemiológica era diligenciada depois da inspeção sanitária realizada pelos funcionários do INVIMA. As principais causas e variáveis epidemiológicas associadas ao confisco de vísceras vermelhas foram estimadas a partir da análise de freqüência, e o valor de perdas econômicas, através de uma análise no mercado nacional. Foram abatidos 8.671 bovinos e 226 búfalos, nos quais 15,8% e 26,5% tiveram confiscos de vísceras vermelhas, respectivamente. Em bovinos, as principais vísceras confiscadas, foram pulmões, fígado e baço com percentagens de 87,4%, 11,1% e 0,6%, respectivamente. Nos búfalos, foram pulmões, fígado e rim com porcentagens de 88,3%, 8,3% e 1,7%, respectivamente. Para as duas espécies, broncoaspiração (86,6%) e enfisema (9,4%) foram as principais causas de confiscos dos pulmões. Para o fígado, foram os abcessos (40,5%) e fasciolose (28,8%) em bois, esteatose hepática (80,0%) e cirrose (20,0%) em búfalos. No caso dos bois, houve diferenças significativas na proporção de animais com fasciolose nos diferentes municípios do Santander (χ² = 50,03, P <0,05). As maiores perdas econômicas durante o período de estudo correspondem ao confisco de pulmão e fígado com valores de 4886 e 2639 USD. Em conclusão, o pulmão foi a principal víscera confiscada em ambas espécies, principalmente associados a broncoaspiração. Estratégias futuras de intervenção, com o objetivo de reduzir a broncoaspiração, e interromper o ciclo de vida da Fasciola hepatica em alguns municípios do Santander são necessários para reduzir as perdas econômicas na cadeia de produção.

Eco-epidemiological study reveals the importance of Triatoma dimidiata in the Trypanosoma cruzi transmission, in a municipality certified without transmission by Rhodnius prolixus in Colombia.

Boyaca department is an endemic area for Chagas disease in Colombia, where 24 of its municipalities have been certified by the PAHO with interruption of Trypanosoma cruzi transmission by R. prolixus. However, the presence of secondary vectors, represent a risk of parasite transmission for citizens and a challenge for the health care institutions. The aim of this work was to investigate eco-epidemiological features of Chagas disease in the municipality of Socota (Boyaca), in order to improve control and surveillance strategies. To understand the transmission dynamics of T. cruzi in this area, we designed a comprehensive, multi-faceted study including: (i) entomological survey in five villages (La Vega, Comaita, Chusvita, Guaquira and Pueblo Nuevo), (ii) blood meal source determination, (iii) T. cruzi infection rate in collected triatomines, (iv) identification of circulating T. cruzi genotypes, (v) serological determination of T. cruzi infection in domestic dogs; and (vi) evaluation of infection in synanthropic mammals. A total of 90 T. dimidiata were collected, of which 73.3% (66/90) and 24.4% (22/90) were collected inside dwellings and peridomestic areas, respectively, while the rest (2/90) in Chusvita Elementary School. T. cruzi infection was evidenced in 40% (36/90) of triatomine bugs using PCR analyses, in which only DTU I was found, and TcI Dom was the most distributed. Blood-meal analysis showed that T. dimidiata only fed of humans. Seroprevalence in domestic dogs was 4.6% (3/66), while that two Didelphis marsupialis captured showed no infection. In conclusion, the high dispersion and colonization of T. dimidiata shown in this municipality, along the high rate of T. cruzi (TcI) infection and its anthropophilic behavior constitute a risk situation for Chagas disease transmission in this municipality certified without R. prolixus. The epidemiological implications of these findings are herein discussed.

Evaluation of an alternative indirect-ELISA test using in vitro-propagated Trypanosoma brucei brucei whole cell lysate as antigen for the detection of anti-Trypanosoma evansi IgG in Colombian livestock.

Surra is a zoonotic disease caused by Trypanosoma evansi, affecting the health and production of the livestock significantly. There are several methods to diagnose this disease, which have different principles, sensitivity, and specificity. Among them, the serological techniques using T. evansi as antigen are powerful tools for its epidemiological surveillance. However, they are poorly used due to inefficient in vitro propagation of T. evansi, which requires the use of laboratory animals for antigen production. In the present study, whole cell lysate of T. brucei brucei propagated in vitro was used as an antigen for the detection of anti-T. evansi immunoglobulin G in cattle through an indirect-ELISA. Based on a total of 45 samples from non-infected and 45 samples from T. evansi infected cattle, the sensitivity and specificity were estimated as 100% and 97.7%, respectively. After the validation, serological and molecular surveys were carried out in 710 cattle samples from two endemic Colombian regions (Antioquia and Arauca departments) for T. evansi where molecular prevalences of ˜7.0% were detected through the year and sporadic outbreaks of T. vivax infections have been associated to low prevalence of this species (<1%). A total of 424 (59.7%) samples were positive by indirect-ELISA T. b. brucei, while PCR test for T. evansi and T. vivax, showed 49 (6.9%) and no positive samples, respectively. Interestingly, categories of animals aged>1 year, Bos taurus breed, and those raised under intensive farming system exhibited a higher seroprevalence to T. evansi (P < 0.05). The results displayed a new alternative for antibody detection anti-T. evansi in livestock, using parasites propagated in vitro as antigen, which presents the advantage of higher standardization potential, and avoid the use of live animal for antigen production. A larger availability of this ELISA will generate useful information for a better understanding of the epidemiologic aspects, as well as for the management and control of these diseases in Colombia. However, the ability of the test to detect and/or cross react with T. vivax infections remains to be investigated.

Molecular surveillance and phylogenetic traits of Babesia bigemina and Babesia bovis in cattle (Bos taurus) and water buffaloes (Bubalus bubalis) from Colombia.

BACKGROUND: Babesia bigemina and B. bovis are two economically important hemoparasites affecting both cattle and buffaloes involved in dairy and beef production. In Colombia, although some parasitological and serological studies suggest an endemicity of these pathogens in areas under 1000 m, little is known about its molecular prevalence in different host. The objective of this study was to estimate the prevalence and molecular traits of these parasites in cattle and buffaloes from two Colombian regions. METHODS: Between 2014 and 2016, a three-point longitudinal survey was designed in farms from Caribbean and Orinoquia regions to evaluate the molecular prevalence of B. bigemina and B. bovis using a nested PCR (n-PCR) targeting hypothetical protein (hyp) and rhoptry-associated protein (RAP-1) genes, respectively. A total of 1432 cattle, 152 buffalo and 1439 Rhipicephalus microplus samples were analyzed. Moreover, phylogenetic relationship of isolates was analyzed using the 18S rRNA gene. RESULTS: A molecular prevalence of 31.6% (24.2% for B. bigemina and 14.4% for B. bovis), 23.6% (6.5% for B. bigemina and 17.7% for B. bovis) and 4.3% (3.5% for B. bigemina and 1.0% for B. bovis) was observed in cattle, buffaloes and Rhipicephalus microplus, respectively. Higher values of infection were observed during the wet season and late wet season; nevertheless, other variables such as age, production type, sex, breed and babesiosis control were also significantly associated with infection. Prevalence analysis showed that B. bovis infection was higher in cattle that coexist with buffaloes, when compared to those which did not. For each species, phylogenetic analyses revealed a high genetic diversity of isolates without clusters related to the isolation source. CONCLUSIONS: To our knowledge, this is the first longitudinal survey that evaluates through molecular methods, the infection of B. bigemina and B. bovis in two important livestock regions from Colombia. This study reveals that the prevalence of infection by Babesia spp., in cattle and buffaloes are modulated by seasonal variations, host factors and vector traits. Our results provide new insights on the epidemiological aspects of infection of Babesia spp., in cattle and buffaloes, which must be taken into consideration when babesiosis control programs are implemented in the study area.

Genetic, host and environmental factors associated with a high prevalence of Anaplasma marginale.

Anaplasma marginale is the most prevalent vector-borne pathogen in the livestock industry in Colombia, causing economic losses of approximately USD 4.2 million per year. The present study reports the seasonal transmission patterns, genetic diversity and phylogeographic traits of A. marginale strains in cattle and buffaloes from Colombian livestock areas. A three-point longitudinal survey was designed to evaluate the above characteristics of farms in the Caribbean and Orinoquía regions. The A. marginale prevalence was evaluated in 1432 cattle blood samples, 152 buffalo blood samples and the hemolymph of 439 ticks using semi-nested PCR (sn-PCR) targeting the msp5 gene. The molecular prevalence in cattle and buffaloes was 54.8% and 13.1%, respectively, with higher values during the wet and late wet seasons. Factors such as age and production system were significantly associated with the infection. Rhipicephalus microplus was the only carrier of A. marginale DNA, with an infection rate of 17.2%. On the other hand, the tandem repeat and microsatellite analyses of the msp1α gene showed high genetic diversity and new tandem repeats that suggested strain adaptation to different transmission modes. Phylogeographic analysis using the msp4 gene showed a relationship between Colombian isolates and Mexican, Brazilian, Venezuelan, European and Asian isolates, as well as two worldwide haplogroups that were associated with the geographical origin of each isolate. In conclusion, this study shows that A. marginale occurs under enzootic stability in both hosts, with a high prevalence of infection during wet months and in animals dedicated to beef production. The genetic variability analyses suggest that a high strain diversity is associated with multiple selective pressures in the study area, while phylogeographic traits suggest a high genetic similarity between Mexican and South American strains.

Spatial-temporal and phylogeographic characterization of Trypanosoma spp. in cattle (Bos taurus) and buffaloes (Bubalus bubalis) reveals transmission dynamics of these parasites in Colombia.

Animal Trypanosomiasis (AT) is one of the most important problems in the Colombian livestock industry reducing its production around 30%. Caribbean and Orinoquia regions play a significant role in the development of this industry, having about 6.9 million cattle and 113,000 buffaloes. Considering the paucity in studies to understand the epidemiological features and control of AT in Colombia, the present study reports the seasonal transmission patterns and phylogeographic traits of the causal agents of AT in cattle and buffaloes from these regions. Between 2014 and 2016, a three-point longitudinal survey was designed to evaluate the mentioned characteristics. Molecular analysis in cattle showed an AT prevalence of 39.2% (T. theileri 38.6%, T. evansi 6.7% and T. vivax 0.2%), with higher values during wet and late wet seasons, while in buffaloes the prevalence was 28.2% (T. theileri 28.2% and T. evansi 1.3%), with higher values during the dry season. Additionally, variables such as tabanid abundance, vector control, breeding system, age and anemia signs were significantly associated with AT prevalence (P<0.05). Only T. theileri infection was higher in cattle with anemia signs than those with normal packed cell volume. Finally, phylogeographic analysis revealed that Colombian T. theileri isolates were associated to specific host genotypes IA and IIB, described worldwide; T. vivax isolates were related to the genotype from West Africa; while T. evansi isolates are related to the South American genotypes and to new genotypes. This is the first longitudinal survey that evaluates through molecular methods, the infection of Trypanosoma spp. in two important livestock regions from Colombia, showing that the clinical effects and prevalence of these trypanosomes in cattle and buffaloes are modulated by seasonal variations, host factors, and parasite traits. The results suggest that these factors have to be taken into account to successfully control AT in these regions.

Molecular and serological detection of Trypanosoma cruzi in dogs (Canis lupus familiaris) suggests potential transmission risk in areas of recent acute Chagas disease outbreaks in Colombia.

Chagas disease is a zoonotic infection widely distributed in tropical and subtropical regions of America, including more than 50% of the Colombian territory. In the last years, an increase of outbreaks of acute Chagas disease has been observed in the east of the country due to environmental changes and mammal movements toward human settlements. Given the importance of dogs (Canis lupus familiaris) as reservoir hosts and sentinels of Trypanosoma cruzi infection across different regions of America, in this study we reported a serological and molecular detection of T. cruzi infection in 242 dogs from an endemic area of Meta department (East of Colombia), with recent emergence of acute Chagas disease outbreaks. The distribution of T. cruzi infection in dogs was not homogeneous, ranging from 0-41.4% and 0-5.1% in different sampling sectors, through serological (ELISA/IFAT) and molecular methods (conventional and real time PCR), respectively. Statistical analysis indicated that dog infection was associated with specific sampling sectors. Our results show a moderate seroprevalence of infection and active circulation of T. cruzi in dogs from this zone, which suggest areas with potential risk of infection to human that must be taken into consideration when Chagas disease control programs need to be implemented.