Strong Evidence of the Role of Donkeys in the Epidemiology of Leptospira spp. in Semiarid Conditions.

Donkeys (Equus asinus) are historically known for their close relationship to humanity, which raises the need to study zoonotic diseases that affect them. In this perspective, leptospirosis stands out as a disease with an economic and public health impact, and its occurrence is facilitated in times of higher rainfall indexes, especially in large urban centers. In view of the scarcity of information about leptospirosis in donkeys, the objective of this study was to detect the presence of Leptospira spp. and anti-leptospiral antibodies in donkeys rescued by a zoonosis center located in the Caatiga biome, Brazilian semiarid region. Overall, 30 donkeys of both sexes, aged between 4 months and 15 years, were used, from which 64 serum samples were collected and submitted to the microscopic agglutination test (MAT). In addition, 64 samples of urine, vaginal and preputial fluid, in duplicates, were subjected to the polymerase chain reaction (PCR) and microbiological. Sixteen (53.3%) animals tested positive in at least one diagnostic test, 12 (40%) of which were positive at MAT and seven (23.3%) in the molecular and bacteriological detection (urine, vaginal, and preputial fluid samples). This is the first report identifying donkeys infected with Leptospira spp. by molecular and bacteriological diagnosis in Brazil, and the first in the world to detect this agent in their genital fluids. The study also shows that donkeys are commonly exposed to leptospires in the Caatinga biome, and this constitutes a One Health-based concern, demonstrating the importance of broad studies where large numbers of humans and animals coexist when investigating zoonotic infections and when planning and implementing control measures for donkeys-associated leptospirosis.

Bovine Leptospirosis in Caatinga Biome, Brazil: New Insights into Diagnosis and Epidemiology.

Bovine leptospirosis causes economic losses and raises public health concerns. It is possible that there are peculiarities in the epidemiology of leptospirosis in regions with a semiarid climate, such as the Caatinga biome in Brazil, where the climate is hot and dry, and the etiological agent require alternative routes of transmission. This study aimed to close knowledge gaps to the diagnosis and epidemiology of Leptospira spp. infection in cows from the Caatinga biome, Brazil. Samples of the blood, urinary tract (urine, bladder and kidney) and reproductive tract (vaginal fluid, uterus, uterine tube, ovary and placenta) were collected from 42 slaughtered cows. Diagnostic tests included were the microscopic agglutination test (MAT), polymerase chain reaction (PCR) and bacterial isolation. Anti-Leptospira spp. antibodies were found in 27 (64.3%) of the animals analyzed using MAT at a 1:50 dilution (cut-off 50), while 31 (73.8%) animals had at least one organ/fluid where the presence of Leptospira spp. DNA was identified, and 29 animals (69%) were positive at bacteriological culture. The highest sensitivity values for MAT were obtained at the cut-off point of 50. In conclusion, even under hot and dry climate conditions, it is possible that Leptospira spp. can spread through alternative routes such as venereal transmission; moreover, a cut-off of 50 is recommended for the serological diagnosis of cattle from the Caatinga biome.

Clinical, histopathological, and molecular findings for Mycobacterium avium subspecies paratuberculosis (MAP) in dairy goats under semiarid conditions.

The objective of this study was to report the occurrence of Mycobacterium avium subspecies paratuberculosis (MAP) in dairy goats, via description of their clinical presentation, histopathological findings, and molecular identification of the infectious agent. Screening was performed using IS900 real-time PCR (qPCR) in milk samples from 179 properties in the semiarid of Northeast region of Brazil. Pooled milk samples from all lactating goats from processing plants were submitted to molecular diagnosis. One property had a positive result at qPCR. The production unit which had the positive sample for MAP was located, and an on-site visit to this property was performed to collect individual milk samples, seven of which tested MAP positive by IS900 qPCR. With permission from the owner, two goats (Animal 1 was positive and Animal 2 was negative on first qPCR for MAP) were acquired and euthanized. Animals 1 and 2 had milk and portions of the duodenum, ileum, colon, and mesenteric lymph nodes positive at qPCR for MAP. Animal 1 also had MAP DNA detected in part of the jejunum and cecum. In animal 2, the ileocecal valve tested positive. MAP was not detected in the blood or feces of either animal; however; it was confirmed for the association of clinical findings, histopathology, and qPCR. The gene IS900 from the positive samples were sequenced and showed a 99% similarity with MAP. The MAP was identified for the first time in the goat milk and tissues in the semiarid region of Northeast Brazil.

Molecular detection of Trypanosoma cruzi in equids in a semiarid zone of Pernambuco - Brazil.

Trypanosoma cruzi is the etiological agent of Chagas disease, a neglected and frequently occurring zoonosis in Central and South American countries. Wild mammals and domestic dogs are the main reservoirs of the parasite in the wild and domestic cycles, respectively. The vectors have a wide variety of food sources that can influence transmission cycles. The aim of this study was to determine the prevalence of T. cruzi infection in donkeys (Equus asininos) and mules (Equus mulus) living in rural areas of the Brazilian semi-arid region. Whole-blood samples from 72 equids (65 donkeys and 7 mules) were analyzed by nested polymerase chain reaction (nested PCR). A total of 51.39% of the samples (37/72) were positive. Phylogenetic analysis identified discrete typing units TcI and TcII, which suggested the possibility that donkeys and mules might be participating in domestic/peridomestic and wild transmission cycles. This was the first report of T. cruzi infection in donkeys and mules in Brazil, with high prevalence of positive animals. This places these animals as potential reservoirs for the parasite and the particular features of these hosts, the presence of vectors and the socioeconomic characteristics of the population under semiarid conditions create interactions that may favor transmission and overlapping T. cruzi infection cycles.

Lack of serological and molecular evidences of Zika virus circulation in non-human primates in three states from Brazil.

BACKGROUND: Zika virus (ZIKV) was discovered in 1947 with the virus isolation from Rhesus monkey (Macaca mulatta) in Uganda forest, Africa. Old World Primates are involved in a sylvatic cycle of maintenance of this arbovirus, however a limited knowledge about the role of New World primates in ZIKV transmission cycles has been established. OBJECTIVE: This work aimed to investigate the presence of enzootic circulation of ZIKV in New World Primates from three Brazilian states: São Paulo, Paraíba, and Paraná. METHODS: We analyzed 100 non-human primate samples collected in 2018 and 2020 from free-ranging and captive environments from São Paulo (six municipalities belonging to Sorocaba region), Paraíba (João Pessoa municipality), and Paraná (Foz do Iguaçu municipality) using reverse transcriptase quantitative polymerase reaction (RT-qPCR) assays, indirect enzyme-linked immunosorbent assay (ELISA), and plaque reduction neutralization test (PRNT). FINDINGS: All samples (n = 141) tested negative for the presence of ZIKV genome from tissue and blood samples. In addition, all sera (n = 58) from Foz do Iguaçu' non-human primates (NHPs) were negative in serological assays. MAIN CONCLUSION: No evidence of ZIKV circulation (molecular and serological) was found in neotropical primates. In addition, the absence of antibodies against ZIKV suggests the absence of previous viral exposure of NHPs from Foz do Iguaçu-PR.

New insights on Leptospira sp. infection in ewes maintained in field semiarid conditions.

Leptospirosis is poorly studied in small ruminants raised in field semiarid conditions. In this study we compared serological, bacteriological and molecular diagnostic methods in ewes maintained in field Brazilian semiarid conditions. Blood, vaginal fluid and urine samples were collected from 60 Morada Nova ewes raised in a semi-intensive system in the Brazilian semiarid. Diagnostic tests performed were microscopic agglutination test (MAT), polymerase chain reaction (PCR) and bacterial isolation. Anti-Leptospira sp. antibodies were found in eight (13.33%) animals analyzed by MAT at reciprocal titer 25 (cut-off 25), while Leptospira sp. DNA was detected in urine or vaginal fluid of 56 animals (93.33%). There was growth of leptospires in 10 urine cultures and in 11 vaginal fluid cultures, however, two of urine (2/60-3.33%) and eight cultures of vaginal fluid (8/60-13.33%) were confirmed by PCR. Two samples of vaginal fluid (one of each animal) were submitted to sequencing demonstrating 99% similarity with L. santarosai and L. interrogans. The highest MAT sensitivities were obtained with reciprocal titer 25 (cut-off 25) compared to 50 and 100. The performance of different diagnostic techniques for leptospirosis in ewes raised in field semiarid conditions allowed a better evaluation of the herd, as well as made it possible to identify carrier animals. Genital route may be important for efficient transmission and without dependence on environmental factors in ewes from semiarid, as well as it's highlighted that titer 1:25 in serology was more efficient, indicating its use in ewes in field semiarid conditions.

Global Prevalence of RNA-Positive Horses for Hepacivirus (EqHV): Systematic Review and Meta-Analysis.

Equine hepacivirus (EqHV) belongs to the Flaviviridae family, genus Hepacivirus and has the greatest genomic identity with the hepatitis C virus (HCV), one of the main causes of chronic liver disease in humans. Due to the limited applicability of studies of HCV in animal hosts, the interest in studies of characterization of viral homologues has been growing. For this reason, we performed a systematic review of the literature with meta-analysis of the prevalence of EqHV and genetic sequencing studies. Twenty-three studies from four different continents were analyzed. The OR meta-analysis (0.98; 95% CI = 0.69-1.39) showed no influence of sex (female or male) on the risk of infection. Variables associated with EqHV infection were indirectly related to animal management such as transport, reproductive practices, among others. The combined prevalence of positive animals was 7.88% (95% CI = 5.23-11.69%), with the highest proportions in Asia (16.13%; 95% CI = 7.79-30.43%), followed by South America (12.03%; 95% CI = 9.58-15.01%), Africa (8.69%; 95% CI = 6.71-11.20%), and Europe (3.63%; 95% CI = 2.10-6.22%). However, these results represent the regional stratification of the epidemiological studies for EqHV published to date and, therefore, cannot extrapolate to determine the continental prevalence of EqHV. It is therefore important to update the systematic review as further research becomes available.

Lack of serological and molecular evidences of Zika virus circulation in non-human primates in three states from Brazil

BACKGROUND Zika virus (ZIKV) was discovered in 1947 with the virus isolation from Rhesus monkey (Macaca mulatta) in Uganda forest, Africa. Old World Primates are involved in a sylvatic cycle of maintenance of this arbovirus, however a limited knowledge about the role of New World primates in ZIKV transmission cycles has been established. OBJECTIVE This work aimed to investigate the presence of enzootic circulation of ZIKV in New World Primates from three Brazilian states: São Paulo, Paraíba, and Paraná. METHODS We analyzed 100 non-human primate samples collected in 2018 and 2020 from free-ranging and captive environments from São Paulo (six municipalities belonging to Sorocaba region), Paraíba (João Pessoa municipality), and Paraná (Foz do Iguaçu municipality) using reverse transcriptase quantitative polymerase reaction (RT-qPCR) assays, indirect enzyme-linked immunosorbent assay (ELISA), and plaque reduction neutralization test (PRNT). FINDINGS All samples (n = 141) tested negative for the presence of ZIKV genome from tissue and blood samples. In addition, all sera (n = 58) from Foz do Iguaçu' non-human primates (NHPs) were negative in serological assays. MAIN CONCLUSION No evidence of ZIKV circulation (molecular and serological) was found in neotropical primates. In addition, the absence of antibodies against ZIKV suggests the absence of previous viral exposure of NHPs from Foz do Iguaçu-PR.

Molecular detection of Leishmania infantum in donkeys and mules under semiarid conditions in Brazil.

Visceral leishmaniasis is a parasitic zoonosis that mainly affects poorest and most vulnerable populations, and domestic dogs are considered to be the main source of infection to the vector and therefore humans. However, several studies have investigated the role of other vertebrate hosts in the disease cycle. In this context, the aim of the present study was to conduct a survey of Leishmania infantum infection in donkeys and mules living in a semiarid region of Brazil. Whole blood sampled from 72 equids (65 donkeys and 7 mules) was used to perform molecular diagnosis using the real-time polymerase chain reaction (qPCR) technique. A total of 25% of the samples (18/72) were positive through qPCR, but there were no significant differences between the species (donkeys or mules), sex (male or female) and abandonment situation of the animals (yes or no). Donkeys and mules living under semiarid conditions have high frequency of L. infantum infection. It is therefore worth assigning importance to these species in the epidemiological cycle of visceral leishmaniasis, either as potential reservoirs or just as an abundant food source for vectors.

Factors associated with Leishmania infection in dogs and geospatial analysis in the Sertão of Paraíba, Northeast Brazil.

Visceral leishmaniasis (VL) is a zoonosis with a worldwide distribution that has a major impact on public health. The aim of this study was to verify the prevalence of canine infection by Leishmania infantum, the factors associated with the infection and its spatial distribution in the municipality of Mãe D'Água, in the Sertão region of Paraíba State, Northeast Brazil. Blood samples were collected from 150 dogs for diagnosis by the DPP®, ELISA-S7®, ELISA-EIE® and qPCR assays. The prevalence was calculated considering the positivity in at least two tests. SaTScan® was used for spatial analysis. The prevalence of canine infection with Leishmania was 18.6% (28/150), with the rural area being identified as a risk factor (Odds Ratio (OR) = 2.93). The permanence of the dog loose during the night (OR = 0.33) and deworming (OR = 0.30) were identified as protective factors. A risk cluster was formed in the northern region of the urban area. Mãe D'Água showed a pattern of active transmission in the rural area, but VL control measures also need to be carried out in the urban area to prevent human cases and the spread of the disease in the risk zone.

High herd-level seroprevalence and associated factors for bovine leukemia virus in the semi-arid Paraíba state, Northeast Region of Brazil.

Epidemiological surveys that investigate infectious diseases such as enzootic bovine leukosis (EBL) are important. Furthermore, estimating the prevalence of such infectious diseases and associated factors is key to assess the disease impact and design control programs. In this study, we identified a high herd-level seroprevalence of EBL in cattle from the semi-arid Paraíba state, Northeast Region of Brazil, using a planned cross-sectional survey. Herd-level and animal-level prevalence were estimated using a two-stage random sampling survey. In total, 2067 cows aged ≥24 months from 400 herds were sampled. An enzyme-linked immunosorbent assay was used to detect specific antibodies directed to the bovine leukosis virus gp51 antigen in both individual and pooled sera. The herd-level and animal-level prevalence was 23.4 % (95 % CI = 19.2-28.1 %) and 10.8 % (95 % CI = 7.5 %-15.3 %), respectively. There were no significant clusters of positive herds (within a radius of 2 km). The factors associated with herd-level prevalence were the exclusive use of hand milking (prevalence ratio [PR] = 1.88), herd size (PR = 1.005), artificial insemination (PR = 2.03), purchase of animals in the previous year (PR = 1.87), and peri-urban farms (PR = 2.09). Prevention measures should be applied at the herd-level, particularly for farms located in peri-urban areas, focusing on good hygiene in hand milking, robust practices and standards for artificial insemination, and serological testing of animals prior to purchase.

Antibody detection and molecular analysis for Mycobacterium avium subspecies paratuberculosis (MAP) in goat milk: Systematic review and meta-analysis.

Paratuberculosis is an incurable infectious disease that affects several species, including goat (Capra hircus). The etiologic agent is Mycobacterium avium subspecies paratuberculosis (MAP) that has tropism for the intestine, causing anorexia, progressive weight loss and death. In goats, the main transmission route is the ingestion of water and food contaminated by infected feces. Affected animals also eliminate the agent through milk, with a potential biological risk to public health. Thus, the aim of this study was to conduct a research of the literature available in electronic media for a systematic review, followed by a meta-analysis of the results found on prevalence and diagnostic tests adopted in the detection of MAP antibodies and DNA in goat milk. The following search parameters were used: "Mycobacterium avium subsp. paratuberculosis" AND (goat OR small ruminant) AND (milk OR pasteurized milk). Strictly obeying pre-established criteria, 437 articles were selected from the respective electronic databases of scientific content: ScienceDirect (285), PubMed (68), Web of Science (60) and Scopus (24), of which nine papers were elected to the construction of the systematic review and meta-analysis. The prevalence of MAP antibodies in milk detected by milk-ELISA ranged from 1.1 to 67.7% and the prevalence of MAP DNA in goat milk detected by MAP-specific polymerase chain reaction (PCR) ranged from 1.94 to 37.74%. A meta-analysis indicated a combined MAP infection prevalence of 8.24%, but with high heterogeneity among study findings (I2 = 98.7%). The identification of the MAP in goat milk implies the need for surveillance of the agent in order to prevent economic losses and impact on public health.

Serological, molecular and bacteriological approaches for detecting Leptospira sp. carrier rams maintained in semiarid conditions.

Even in the adverse environmental conditions of the semiarid region, leptospires can survive and spread by alternative routes of transmission, such as sexual in ewes, however, there is no data on rams. Thus, the present study aimed to evaluate the use of serological, molecular and microbial tools applied to diagnosis of Leptospira sp. Infection in rams maintained in semiarid conditions. Biological samples of urinary (urine, kidney and bladder) and genital (vas deferens, epididymis tail and vesicular gland) tracts were collected from 40 slaughtered rams for polymerase chain reaction (PCR) and bacterial isolation, as well as blood samples for antibody detection through microscopic serum agglutination test (MAT). Anti-Leptospira antibodies were found in five (12.5%) animals with antibody titer of 50 and 2 (5%) for the titer 100 for serogroups Pyrogenes, Ballum, Icterohaemorrhagiae and Australis. Leptospira sp. DNA was found in PCR of organs and urine of 30 (75%) animals. Overall, 240 fragments of organs from the urinary and genital tracts and urine were evaluated, with 93 (38.7%) positive samples, being 48/120 (40%) for the urinary tract and 45/120 (37.5%) for the genital. There was no statistically significant difference between the tracts. A bladder sample was sent for sequencing and showed 99% similarity with L. interrogans. Of the 240 cultures evaluated, 59 (24.5%) had leptospire growth, being that 23 (39%) were confirmed in PCR. Considering the PCR of organs and urine and bacterial growth as gold standards, the cut-off 50 in MAT showed greater sensitivity when compared to cut-off 100, regardless of the material used. The great proportion of leptospiral DNA in organs, urine and culture and bacterial growth from the genital tracts reinforce its importance as an extra-renal site and highlights the possible role of rams in venereal transmission, as well as the sensitivity of the cut-off 50 suggested its adoption in the serology of rams maintained in semiarid conditions.

Molecular detection of Leishmania infantum in donkeys and mules under semiarid conditions in Brazil / Detecção molecular de Leishmania infantum em asininos e muares sob condições semiáridas

Abstract Visceral leishmaniasis is a parasitic zoonosis that mainly affects poorest and most vulnerable populations, and domestic dogs are considered to be the main source of infection to the vector and therefore humans. However, several studies have investigated the role of other vertebrate hosts in the disease cycle. In this context, the aim of the present study was to conduct a survey of Leishmania infantum infection in donkeys and mules living in a semiarid region of Brazil. Whole blood sampled from 72 equids (65 donkeys and 7 mules) was used to perform molecular diagnosis using the real-time polymerase chain reaction (qPCR) technique. A total of 25% of the samples (18/72) were positive through qPCR, but there were no significant differences between the species (donkeys or mules), sex (male or female) and abandonment situation of the animals (yes or no). Donkeys and mules living under semiarid conditions have high frequency of L. infantum infection. It is therefore worth assigning importance to these species in the epidemiological cycle of visceral leishmaniasis, either as potential reservoirs or just as an abundant food source for vectors.

Resumo A leishmaniose visceral é uma zoonose parasitária que afeta principalmente populações mais pobres e vulneráveis, e os cães domésticos são considerados as principais fontes de infecção para o vetor e, portanto, para os humanos. Porém diversos estudos têm pesquisado o papel de outros hospedeiros vertebrados no ciclo da doença. Neste contexto, objetivou-se realizar um levantamento da infecção por Leishmania infantum em asininos e muares, vivendo em região semiárida do Brasil. Foi utilizado sangue total de 72 equídeos (65 asininos e 7 muares) para a realização de diagnóstico molecular por meio da técnica de Reação em Cadeia de Polimerase em Tempo Real (qPCR). Um total de 25% das amostras (18/72) resultaram positivas na qPCR, porém não houve diferença significativa entre as espécies (asininos e muares), sexo (macho e fêmea) e situação de abandono dos animais (sim ou não). Asininos e muares, vivendo em condições semiáridas, apresentam alta frequência de infecção por L. infantum, sendo válido atribuir importância a essas espécies no ciclo epidemiológico da leishmaniose visceral, seja como um reservatório em potencial, seja apenas como uma fonte alimentar abundante para os vetores.

True prevalence and spatial distribution of equine infectious anemia virus (EIAV) in horses from northeast region of Brazil

Equine infectious anemia (EIA) is a viral infection, caused by a lentivirus of the Retroviridae family, Orthoretrovirinael subfamily and its occurrence generates significant economic losses due to culling of positive animals as a measure of infection control. The objective of this work was to determine the prevalence of horses positive for equine infectious anemia virus (EIAV) and to identify the occurrence of areas with higher densities of cases in the states of Paraíba (PB), Pernambuco (PE), Rio Grande do Norte (RN) and Ceará (CE), Northeast region of Brazil, during the rainy (May and June) and dry (October and November) periods of 2017 and 2018. Serum samples from 6,566 horses from the states of PB, PE, RN and CE, Brazil, provided by the Laboratório Veterinária Diagnóstico - Ltda., were used. Serological diagnosis of EIA was performed using indirect enzyme-linked immunosorbent assay (ELISA) as a screening test and agar gel immunodiffusion test (AGID) as a confirmatory test. The apparent prevalence was obtained by dividing the number of seroreactive animals by the total number of animals, while the true prevalence was estimated by adjusting the apparent prevalence, considering the sensitivity (100%) and specificity (98.6%) of the diagnostic protocol used. For the construction of Kernel estimates, the Quartic function was used. In the dry season, of the 1,564 animals sampled, 28 were serologically positive, of which 19 belonged to the state of Ceará, 7 to Paraíba and 2 to Rio Grande do Norte. In 2018, it was observed that, during the rainy season, 26 of the 1,635 horses were seroreactive, with 19 cases resulting from Ceará, 4 from Paraíba and 3 from Pernambuco. In the dry season, 32 of the 1,526 animals were seroreactive to EIAV, of which 26 were from Ceará, 3 from Paraíba, 1 from Rio Grande do Norte and 2 from Pernambuco. In the dry period of 2017, the CE had a real prevalence of 1.22% (95% CI = 0.05 - 2.99%). In 2018, during the rainy season, prevalences of 0.03% (95% CI = 0 - 1.18%) were identified in CE and 1.69% (95% CI = 0 - 8.38%) in PE. Regarding the 2018 dry period, a prevalence of 1.32% (95% CI = 0.26 - 2.84%) was found in the state of CE. In both dry and rainy periods of 2017, the presence of spatial clusters of animals positive for EIA was observed, mainly in the border areas among the states of CE, PE, PB and RN. In 2018, there was a variation in the distribution of areas with higher densities of cases between the rainy and dry periods. The state of CE had the highest prevalence of positive animals and the presence of areas with higher densities of EIA cases in both climatic periods, in the years 2017 and 2018. In some municipalities of the CE, important sporting events of agglomeration of animals take place, which can favor the transmission of EIAV by facilitating the contact of infected and susceptible animals. Population density may be a factor associated with the higher prevalence observed in this region, as it has the second largest herd among the states studied. Higher densities indirectly contribute to the occurrence of infectious diseases, as they favor the contact of infected and susceptible animals. The occurrence of higher densities of cases in the border areas of the states of PE, RN, CE, and PB may be related to the greater movement of animals in these regions, favoring the indirect contact of infected horses with susceptible ones. The observed results demonstrate the circulation of the EIAV in four states in the Northeast region of Brazil.(AU)

Didelphis albiventris as a carrier of Leptospira sp. in the central nervous tissue in the semiarid region of Northeast, Brazil.

Leptospirosis has been investigated in several species of wild animals. The white-eared opossum (Didelphis albiventris) is a mammal common in the brazilian semi-arid, so, this study aimed to investigate its role in the occurrence of the leptospirosis in the region Northeast of Brazil. 12 animals were used, from which samples were collected for the attempt of isolation, molecular detection and serological examination. There was no microbial growth, nor were any anti-Leptospira sp. antibodies found in the serological samples. The PCR detected leptospiric DNA in the central nervous system (CNS) of five animals (41.7 %). The gene in one of the samples was sequenced and showed identity with Leptospira interrogans. The presence of Leptospira sp. in the CNS of Didelphis albiventris does not allow the characterization of the studied animals as reservoirs with potential for transmission of the pathogen in the region, however it represents a site that needs to be further investigated.

Leptospira sp. vertical transmission in ewes maintained in semiarid conditions.

Leptospirosis is a globally distributed disease associated with reproductive failures in livestock; however, its pathogenesis has not been fully elucidated. Results from the present study indicate there is a presence of Leptospira sp. in organs and fluids of fetuses from ewes slaughtered in the semiarid region of Brazil. Twenty-nine fetuses from 23 ewes determined to be Leptospira sp.-positive using PCR were sampled (14 and 15 in dry and rainy seasons, respectively). Fetal samples of blood, central nervous system (CNS), lung, liver, spleen, stomach contents, peritoneal fluid, kidney, bladder, urine and reproductive system were collected. Diagnostic methods included the microscopic agglutination test (MAT), polymerase chain reaction (PCR) and bacterial isolation. Of the 29 fetuses, 24 (82.8 %) had at least one Leptospira sp.-positive organ or fluid, as determined using PCR, and of a total of 209 samples, 62 (29.7 %) contained leptospiral DNA. Of the 99 samples collected during the dry season, 42 (42.4 %) were positive, and of 110 samples collected during the rainy season, 20 (18.2 %) were positive (P = 0.0001). There was deoxyribonucleic acid (DNA) sequencing of three samples of kidney, CNS and liver, and in all of these, there was 99.3 % similarity with Leptospira interrogans. Leptospires were present in cultures of pooled samples from fetuses with deformities. Results indicate there is vertical (maternal-to-fetus) transmission which would represent an alternative transmission route for the spread of Leptospira sp. in ewes, suggesting molecular detection is essential in the investigation of leptospirosis in fetuses to identify animals that have been infected with this bacterium.

Use of serological and molecular techniques for detection of Leptospira sp. carrier sheep under semiarid conditions and the importance of genital transmission route.

It is possible that there are peculiarities in the epidemiology of leptospirosis in regions with a semiarid climate, where the environment is often adverse, allowing the occurrence of alternative transmission routes. The objective of the work was to generate contributions to the diagnosis and epidemiology of Leptospira sp. infection in sheep reared in semiarid conditions, using serological, molecular and microbiological techniques for diagnosis in dry and rainy seasons. Samples of blood, vaginal fluid, urine, bladder, kidney, uterus, uterine tube, ovary and placenta were collected from 104 sheep (52 animals per season - dry and rainy) slaughtered in the Brazilian semiarid. Diagnostic tests performed were microscopic agglutination test (MAT), polymerase chain reaction (PCR) and bacterial isolation. Anti-Leptospira sp. antibodies were found in 26 (25%) of the animals analyzed by MAT at 1:50 dilution (cut-off 50), while 69 (66.3%) animals had at least one organ/fluid with the presence of Leptospira sp. DNA. Overall, PCR was performed on 758 fragments of organs/fluids from the genital and urinary tracts, and 519 (68.5%) samples tested positive. PCR-positivity was statistically different in dry (46.2%) and rainy (11.5%) seasons for vaginal fluid. It was possible to perform the DNA sequencing in nine samples with 99% similarity to L. interrogans and recovery of viable strains in three samples of vaginal fluid. Regardless of the biological material used in PCR to detect carrier animals and the season, the highest MAT sensitivity values were obtained with cut-off 50 compared to 100. The results obtained indicate that, even in the adverse environmental conditions of the semiarid region, leptospires may survive and propagate by alternative routes of transmission, such as sexual, and the presence of PCR-positive genital tracts in ewes suggests that sexual transmission may play an important role in the epidemiology of the disease in sheep in Brazilian semiarid. In addition, it is suggested the use of titer 50 as cut-off point at serology in semiarid conditions.

High frequency of seropositive and carriers of Leptospira spp. in pigs in the semiarid region of northeastern Brazil.

Carrier animals are considered key in the transmission cycle of leptospirosis. Although investigations have been carried out on several species, the role of pigs in the epidemiology of the disease is still poorly studied in the semi-arid region. Thus, the objective of this study was to determine the presence of Leptospira spp. in the genitourinary tract of pigs intended for slaughter. Fifty pigs were used: adults and unvaccinated. Samples of the kidney, urine, and vaginal fluid were collected for the molecular detection of Leptospira spp. and blood samples for the serological test. The molecular test was performed using the polymerase chain reaction (PCR), and the serological test was performed with the microscopic agglutination test (MAT). Samples with DNA amplification were submitted to genetic sequencing. Twenty (40%) animals were found with anti-Leptospira spp. antibodies, and the majority of the reactions (50%) occurred for the serogroup Tarassovi. Leptospiral DNA was found in the tissue of 11 (22%) pigs. The gene from a urine sample was sequenced and showed similarity to L. borgpetersenii. The results evidenced a high rate of porcine carriers; therefore, they appear to be important sources of agent infection, being potential transmitters of the disease to other animal species and man.

High proportion of cattle and sheep seropositive and renal carriers of Leptospira sp. under semiarid conditions / Alta proporção de bovinos e ovinos soropositivos e portadores renais de Leptospira sp. em condições semiáridas

The aims of this study were to perform serological and molecular detection of Leptospira sp. infection in cattle and sheep under semiarid conditions. Based on a preliminary study performed in our research group, we selected six rural properties showing a positivity ≥ 60% for Sejroe serogroup with titer ≥ 200 measured in serological tests from cattle. In the present study, blood and urine samples were collected from 99 females of reproductive age (51 cattle and 48 sheep) for serological diagnosis, molecular detection and Leptospira sp. attempt to strain recovery. Of the 99 analyzed animals 38.4% (38/99) were positively reactive at the serological tests. Of them, 49% (25/51) were cattle and 27.1% (13/48) sheep. The serogroups detected in cattle were Sejroe (36.8%), Hebdomadis (26.3%), Australis (10.5%), Djasiman (10.5%), Ballum (5.3%), Pomona (5.3%), and Cynopteri (5.3%) with titers of 100–800. In sheep, the reactive serogroups were Australis (27.3%), Ballum (27.3%), Djasiman (18.1%), Tarassovi (9.1%), Icterohaemorrhagiae (9.1%), and Cynopteri (9.1%) with titers of 100–400. Leptospiral DNA was detected in nine urine samples, including five cattle and four sheep. Property 1 showed the highest serological positivity frequencies for both cattle (70.6%) and sheep (70.6%). Similarly, the highest frequency of DNA detection was also found (eight samples, 89%). In this property, we observed the existence of consorted rearing of cattle and sheep with close coexistence between these species. In semiarid conditions, transmission among animals of the same species seems to be the main form of Leptospira sp. dissemination in cattle and sheep herds. However, the contribution of other domestic and wild animals cannot be discarded. The practice of consorted rearing of cattle and sheep and their close coexistence may facilitate the spread of the pathogen in rural properties.

Os objetivos deste estudo foram realizar detecção sorológica e molecular da infecção por Leptospira sp. em bovinos e ovinos em condições semiáridas. Com base em estudo preliminar realizado em nosso grupo de pesquisa, foram selecionadas seis propriedades rurais com soropositividade ≥ 60% para o sorogrupo Sejroe com título ≥ 200 em bovinos. No presente estudo, amostras de sangue e urina foram coletadas de 99 fêmeas em idade reprodutiva (51 bovinos e 48 ovinos) para diagnóstico sorológico, detecção molecular e tentativa de recuperação de estirpesde Leptospira sp. Dos 99 animais analisados, 38,4% (38/99) foram sororeativos nos testes sorológicos. Destes, 49% (25/51) eram bovinos e 27,1% (13/48) ovinos. Os sorogrupos detectados em bovinos foram Sejroe (36,8%), Hebdomadis (26,3%), Australis (10,5%), Djasiman (10,5%), Ballum (5,3%), Pomona (5,3%) e Cynopteri (5,3%) com títulos de 100 a 800. Nos ovinos, os sorogrupos reativos foram Australis (27,3%), Ballum (27,3%), Djasiman (18,1%), Tarassovi (9,1%), Icterohaemorrhagiae (9,1%) e Cynopteri (9,1%) com títulos de 100-400. O DNA leptospiral foi detectado em nove amostras de urina, incluindo cinco bovinos e quatro ovinos. A propriedade 1 apresentou as maiores frequências de positividade sorológica para bovinos (70,6%) e ovinos (70,6%). Da mesma forma, a maior frequência de detecção de DNA também foi encontrada (oito amostras, 89%). Nesta propriedade observou-se a existência de criação consorciada de bovinos e ovinos com estreita convivência entre estas espécies. Em condições semiáridas, a transmissão entre animais da mesma espécie parece ser a principal forma de disseminação de Leptospira sp. em rebanhos bovinos e ovinos. No entanto, a contribuição de outros animais domésticos e selvagens não pode ser descartada. A prática de criação consorciada de bovinos e ovinos e sua estreita convivência podem facilitar a disseminação do patógeno em propriedades rurais.