Who coined the term "One Health"? Cooperation amid the siloization.

This short communication is an effort to describe and elucidate the trajectory of the modern historical concept of "One Health." It is dedicated to the many integrated approaches of health closely related to One Health, while also recognizing the contribution and origination of One Health perspectives/notions from those that have led the way and spearheaded this movement while considering Indigenous cultures across the world. The effects of synergies of those involved in building these integrative approaches are potentially bigger and better lasting than the sum of the individual players. It is only through collaboration, cooperation and diplomacy that we can achieve impactful transformation to benefit health. In this commentary, we aim to appropriately and accurately describe how the current use of "One Health" came to be and who were the main players.

Hemotropic Mycoplasmas (Hemoplasmas) in Free-Ranging Azara's Agoutis (Dasyprocta azarae) from an Urban Area of Southern Brazil.

Hemotropic mycoplasmas (hemoplasmas) are opportunistic bacteria that attach to the erythrocyte surface, causing infectious anemia in several mammalian species, including rodents. Studies surveying native Azara's agoutis (Dasyprocta azarae) in Brazil are lacking. Accordingly, the present study aimed to assess hemoplasmas infection in free-ranging agoutis from an urban environmental conservation area in Curitiba, southern Brazil. Overall, 11/35 (31.43%) agoutis were positive to hemoplasmas by quantitative PCR (cycle threshold≤34.4). Sequencing of the 16S ribosomal RNA gene indicated Mycoplasma haemomuris infection, closely related to M. haemomuris subsp. ratti, suggesting hemoplasma transmission from urban rats to agoutis. Because the main route of M. haemomuris transmission has been direct rodent-to-rodent infection, the relatively lower positivity that we detected may be the result of low intraspecies contact due to the smaller social units of agoutis, generally consisting of two to four individuals, and low interspecies contact due to only sporadic agouti-rat interactions in urban settings, compared with other rodent species interactions. Further studies should be conducted to determine whether the hemoplasma infection that we found can cause clinical onset and life-threatening anemia in agoutis.

Natural SARS-CoV-2 Infection in a Free-Ranging Black-Tailed Marmoset (Mico melanurus) from an Urban Area in Mid-West Brazil.

The emergence of spillover pathogens such as SARS-CoV-2 is a risk to vulnerable human populations. We report natural SARS-CoV-2 infection in a free-ranging adult female black-tailed marmoset (Mico melanurus) from an urban area of Cuiabá, Mato Grosso State, Brazil. The animal was found after a motor vehicle collision without previous clinical history. Necropsy confirmed polytrauma. Severe multifocal to coalescent haemorrhage and mild multifocal peribronchial lymphocytic hyperplasia were seen in lung sections. The alveolar septa were multifocally expanded by a few lymphocytes. Mild lymphocytic periportal hepatitis and interstitial nephritis were found. The lymphoid nodules of the large intestine showed marked lymphocytic hyperplasia. Infection by SARS-CoV-2 was established by viral RNA detection in a pool of nasopharyngeal and oropharyngeal swabs and liver samples. Immunohistochemistry detected the viral nucleocapsid protein in sections of lung, liver, spleen, lymph nodes and large intestine, and spike protein antigen in lung tissue. This is the first report of naturally occurring SARS-CoV-2 infection in a New World monkey. Platyrrhine species should be included as potential hosts of natural infection of SARS-CoV-2.

Leptospira spp. Antibody in Wild Boars (Sus scrofa), Hunting Dogs (Canis lupus familiaris), and Hunters of Brazil.

Hunting activities are a potential risk factor for human infection with Leptospira spp. and, although wild boar seroprevalence has been studied, there are no concurrent serosurveys of wild boars (Sus scrofa), hunting dogs (Canis lupus familiaris), and hunters. The aim of our study was to assess the seroprevalence of Leptospira spp. antibodies in free-ranging wild boars, hunting dogs, and hunters, and risk factors associated with exposure in southern and central-western Brazil. Leptospira spp. antibodies were serologically detected using the microscopic agglutination test, with a total 30 serovars. Overall, 12.2% (9/74) of wild boars and 10.6% (16/170) of hunting dogs were seropositive for at least one serovar and all hunters 0.0% (0/49) were seronegative for Leptospira spp. Seropositivity was statistically higher in 42.1% (8/19) wild boars from natural areas when compared to 2.4% (1/41) from anthropized areas (P<0.001), with prevalence ratio of 17.14 (95% confidence interval: 2.29-128.36). Despite the limited sample size, our findings showed that hunters may be less exposed to Leptospira spp. than are wild boars, particularly in natural areas where Leptospira spp. may be maintained by wild reservoirs. In addition to acting as sentinels, hunting dogs may play a role in disease transmission of sylvatic leptospiral serovars.

Tick-borne pathogens in carthorses from Foz do Iguaçu City, Paraná State, southern Brazil: A tri-border area of Brazil, Paraguay and Argentina.

Tick-borne diseases (TBD) constitute an important group of illness affecting animals and humans worldwide. In Brazil, carthorses are frequently exposed to ticks and tick-borne pathogens, leading to impairment of horse performance and imposing restrictions by the international veterinary authorities for the importation of horses. Accordingly, this study has aimed to i) determine the prevalence of the TBD agents Theileria equi, Babesia caballi, Ehrlichia spp., and hemotropic mycoplasmas in carthorses, ii) identify the tick species parasitizing the animals, and iii) determine factors associated with exposure/infection in Foz do Iguaçu City, Parana state, southern Brazil. A total of 103 carthorses were screened for anti-T. equi and anti-Ehrlichia spp. antibodies by indirect fluorescent antibody assays (IFA). Samples were also tested by PCR assays targeting the 18S rRNA gene of T. equi and B. caballi, and 16S rRNA gene of hemoplasmas. Additionally, PCR assays targeting the 16S rRNA, disulfide bond formation protein (dsb) and tandem repeat proteins 36 (trp36) genes of Ehrlichia spp. were also performed. Antibodies to T. equi and Ehrlichia spp. were detected in 43/103 (41.75%; 95% CI: 32.10-51.88%) and 5/103 (4.85%; 95% CI: 1.59-10.97%) horses by IFA, respectively. DNA of T. equi and B. caballi were found in 25/103 (24.27%; 95% CI: 16.36-33.71%) and 10/103 (9.71%; 95% CI: 4.75-17.13%) carthorses, respectively, and all tested negative for Ehrlichia spp. and hemoplasmas. All sequences showed ≥99% identity with multiple T. equi and B. caballi 18S rRNA gene sequences deposited in GenBank. Overall, 191 Dermacentor nitens ticks were collected from 25/103 (24.27%) animals. Carthorses older than 5 years were more likely to be positive for T. equi (p < 0.05). In conclusion, equine piroplasmosis agents are highly prevalent in carthorses from Foz do Iguaçu City. The low prevalence of Ehrlichia spp. found may be due to the absence of Amblyomma ticks infesting animals, which should be further investigated.

Ticks and serosurvey of anti-Rickettsia spp. antibodies in wild boars (Sus scrofa), hunting dogs and hunters of Brazil.

BACKGROUND: Rickettsia bacteria are responsible for diseases in humans and animals around the world, however few details are available regarding its ecology and circulation among wild animals and human populations at high transmission risk in Brazil. The aim of this study was to investigate the occurrence of ticks and Rickettsia spp. in wild boars, corresponding hunting dogs and hunters. METHODS: Serum samples and ticks were collected from 80 free-range wild boars, 170 hunting dogs and 34 hunters from southern and central-western Brazil, from the Atlantic Forest and Cerrado biomes, respectively, between 2016 and 2018. Serum samples were tested by indirect immunofluorescent-antibody assay (IFA) to detect IgG antibodies against Rickettsia rickettsii, Rickettsia parkeri, Rickettsia bellii, Rickettsia rhipicephali and Rickettsia amblyommatis. Tick species were identified by morphological taxonomic keys, as previously described. A total of 164 ticks including A. sculptum, A. brasiliense and A. aureolatum were tested in PCR assays for Spotted Fever Group (SFG) Rickettsia spp. RESULTS: A total of 58/80 (72.5%) wild boars, 24/170 (14.1%) hunting dogs and 5/34 (14.7%) hunters were positive (titers ≥ 64) to at least one Rickettsia species. A total of 669/1,584 (42.2%) ticks from wild boars were identified as Amblyomma sculptum, 910/1,584 (57.4%) as Amblyomma brasiliense, 4/1,584(0.24%) larvae of Amblyomma spp. and 1/1,584 (0.06%) nymph as Amblyolmma dubitatum. All 9 ticks found on hunting dogs were identified as Amblyomma aureolatum and all 22 ticks on hunters as A. sculptum. No tested tick was positive by standard PCR to SFG Rickettsia spp. CONCLUSIONS: The present study was the concomitant report of wild boar, hunting dog and hunter exposure to SFG rickettsiae agents, performed in two different Brazilian biomes. Wild boar hunting may increase the risk of human exposure and consequently tick-borne disease Wild boars may be carrying and spreading capybara ticks from their original habitats to other ecosystems. Further studies can be required to explore the ability of wild boars to infecting ticks and be part of transmission cycle of Rickettsia spp.

Serosurvey of bluetongue, caprine arthritis-encephalitis (CAE) and Maedi-Visna in Barbary sheep (Ammotragus lervia) of a southern Brazilian zoo / Estudo sorológico dos vírus da língua azul, da artrite-encefalite caprina e Maedi-Visna em aoudads (Ammotragus lervia) em um zoológico do Sul do Brasil

Bluetongue (BT) is an infectious and non-contagious disease of compulsory notification which may affect domestic and wild ruminants, transmitted by Culicoides spp. midges. Despite the high morbidity and mortality in sheep, role of wild animals in the BT cycle remains unclear. Caprine arthritis-encephalitis (CAE) and Maedi-Visna virus (MVV) have been reportedly found in goats and sheep, but not described in wildlife species. Accordingly, serum samples from 17 captive Barbary sheep (Ammotragus lervia) from Curitiba zoo, southern Brazil, were tested for bluetongue, caprine arthritis-encephalitis (CAE) and Maedi-Visna viruses by agar gel immunodiffusion (AGID) and enzyme linked immunosorbent assay (ELISA). Antibodies for bluetongue were observed in 6/17 (35.3%) Barbary sheep by AGID test and in 7/17 (41.2%) by ELISA. All samples were negative for the presence of antibodies against caprine arthritis-encephalitis (CAE) and Maedi-Visna viruses. These findings indicate that Barbary sheep may be infected by bluetongue virus and act as wildlife reservoir in both captive and free-range environments.(AU)

A língua azul é uma doença infecciosa e não contagiosa, de notificação obrigatória, que pode afetar ruminantes domésticos e silvestres, transmitida por mosquitos do gênero Culicoides spp. Apesar da alta morbidade e mortalidade em ovelhas, o papel de animais silvestres no ciclo do vírus da língua azul é desconhecido. A artrite encefalite caprina (CAE) e Maedi-visna vírus (MVV) tem sido encontrados em cabras e ovelhas, porém não há descrição em espécies selvagens. Amostras de soro de 17 aoudads (Ammotragus lervia), mantidos em cativeiro no Zoológico de Curitiba, Sul do Brasil, foram testadas para os vírus da língua azul, da artrite encefalite caprina (CAE) e Maedi-visna, utilizando imunodifusão em gel de ágar e o teste de ELISA (enzyme linked immunosorbent assay). Foram observados anticorpos para o vírus da língua azul em 35,3% (6/17) aoudads utilizando a imunodifusão em gel de ágar e 41,2% (7/17) no ELISA. Todas as amostras foram negativas para a presença de anticorpos contra os vírus da artrite encefalite caprina e Maedi-visna. Esses resultados indicam que os aoudads podem ser infectados pelo vírus da língua azul e atuar como um reservatório silvestre tanto em cativeiro quanto em vida livre.(AU)

Serological survey of anti-Leptospira spp. antibodies in Barbary sheep (Ammotragus lervia) at the Curitiba Zoo, southern Brazil / Estudo sorológico de anticorpos anti-Leptospira spp. em aoudads (Ammotragus lervia) no zoológico de Curitiba, sul do Brasil

Leptospirosis is a worldwide zoonosis, affecting humans, domestic and wild animals. The present study aimed to evaluate prevalence of anti-Leptospira spp. antibodies in Barbary sheep at the Curitiba zoo. Microscopic agglutination test (MAT) was performed using 17 serogroups. Antibodies against Leptospira spp. were observed in 23.5% samples and Icterohaemorrhagiae was the only prevalent serogroup. The presence of anti-Leptospira antibodies in Barbary sheep indicates exposure to leptospires; thus monitoring and preventive measures are necessary in zoo's captive animals, since they can act as sentinels of environmental exposure in an area with high movement of people.(AU)

A leptospirose é uma zoonose mundial que afeta seres humanos, animais domésticos e selvagens. O presente estudo objetivou avaliar a prevalência de anticorpos anti-Leptospira spp. em aoudads do zoológico de Curitiba. Foi realizado o teste de Soroaglutinação microscópica (SAM) utilizando 17 sorogrupos. Anticorpos contra Leptospira spp. foram observados em 23.5% das amostras de aoudads e Icterohaemorrhagiae foi o único sorogrupo prevalente. A presença de anticorpos em aoudads indica exposição a leptospiras portanto monitoramento e medidas preventivas são necessários em animais confinados em zoológicos, uma vez eles podem atuar como sentinelas de exposição ambiental em uma área com alta circulação de pessoas.(AU)

Dog and cat population dynamics in an urban area: evaluation of a birth control strategy / Dinâmica populacional canina e felina em área urbana: avaliação da estratégia de controle reprodutivo

For canine and feline population management in an urban area, a set of well-developed strategies is required to prevent overpopulation, the abandonment of animals, and zoonosis. An understanding of the dynamics of these populations and a characterization of these populations are necessary for action planning. The proposed strategies should be monitored and evaluated so that canine and feline population management programs are properly implemented. Population management programs can be improved through evidence based adaptive management. The objective of this study was to characterize the canine and feline populations and their dynamics in an urban area and to evaluate the impact of a birth control program. Three cross-sectional census surveys and a birth control program were conducted in a neighborhood of São Paulo area with 4,275 households. The two first surveys were performed in 2005 and 2006, prior to implementation of the birth control program, and were used to characterize the canine and feline populations. The third survey was performed in 2008, eighteen months after the birth control strategy had been established. The canine population decreased from 2006 to 2008, after birth control. The mean age for the canine population was 3.36 years; 65% of the dogs were younger than 3 years of age. The mean life expectancy at birth was 3.9 years for male dogs and 5.9 years for female dogs. The mean age for the feline population was 1.66 years; 74% of the cats were 1 year of age or less. The canine and feline populations had a high mortality rate for juveniles younger than 1 year of age. There was an 8% and an 18% decrease in canine and feline birth rates, respectively, after spay or neuter intervention. There was a high animal population turnover, which was more pronounced in the feline population.(AU)

Para o manejo populacional canino e felino em área urbana, um conjunto de estratégias é necessário para evitar a superpopulação, o abandono animal e a transmissão de zoonoses. O entendimento da dinâmica e a caracterização dessas populações são fundamentais para o planejamento das ações, monitoramento e avaliação do programa de manejo populacional e de suas estratégias. Programas de manejo populacional podem ser melhorados por meio de uma gestão adaptativa baseada em evidências. Para avaliar o impacto do controle reprodutivo de cães e gatos foram feitos três estudos transversais por meio de censos em uma área de São Paulo com 4.275 famílias. Os dois primeiros censos foram realizados em 2005 e 2006, antes do controle reprodutivo, e usado para caracterizar as populações canina e felina. O terceiro censo foi realizada em 2008, 18 meses após a estratégia de controle reprodutivo ter iniciado. A população canina diminuiu de 2006 a 2008, após o controle reprodutivo. A idade média para a população canina foi 3,36 anos. A expectativa de vida média ao nascer foi de 3,9 anos para os cães machos e 5,9 anos para as fêmeas. A idade média da população felina foi de 1,66 anos. 65% dos cães eram menores de 3 anos de idade e 74% dos gatos tinham 1 ano de idade ou menos. As populações canina e felina tiveram alta taxa de mortalidade de animais jovens. Houve uma diminuição de 8% e 18% nas taxas de natalidade canina e felina, respectivamente, após a intervenção do controle reprodutivo. Houve uma elevada renovação da população animal, mais pronunciada para a população felina.(AU)

Mycoplasma ovis infection in goat farms from northeastern Brazil.

Although Mycoplasma ovis (formerly Eperythrozoon ovis) has been described in small ruminants worldwide, data on M. ovis in goats remain scarce. Accordingly, the aims of the present study were to i) determine the prevalence of hemoplasmas in goats, ii) identify the tick species parasitizing the animals, and iii) determine factors associated with infection in five dairy and three beef goat farms from the Paraíba State, northeastern Brazil. Blood samples were obtained from 402 goats. Samples were screened for hemoplasmas using a pan-hemoplasma PCR. The positive samples were confirmed by sequencing. An epidemiological questionnaire was given to each farm owner addressing age, gender, and presence of ticks. A total of 158/402 (39.3%) goats were positive for M. ovis by PCR. Sequencing of PCR positive samples has shown ≥99% identity with multiple M. ovis 16S rDNA sequences deposited in GenBank, including M. ovis isolates from humans. Dairy (OR=2.15; 95% CI: 1.40-3.32%; P=0.0004) and anemic goats (OR=2.33; 95% CI: 1.51-3.71%; P=0.0001) were more likely to be infected than beef and non-anemic animals, respectively. Amblyomma parvum (49/52, 94.23%) and Rhipicephalus microplus (3/52, 5.77%) were the tick species found parasitizing the animals, with no significant association between the presence of ticks and infection by M. ovis (P=0.1164). This is the first reportedly molecular detection of M. ovis infection in goats from South America. In conclusion, M. ovis is highly prevalent in goats from northeastern Brazil, mainly in dairy animals.

Molecular screening for hemotropic mycoplasmas in captive Barbary sheep (Ammotragus lervia) in southern Brazil.

AIM: This study is part of an active surveillance program for monitoring animal health status in endangered species, and was conducted to screen captive Barbary sheep (Ammotragus lervia) for hemoplasma infection. MATERIALS AND METHODS: A total of 12 blood samples were collected, DNA extracted and further tested by a pan-hemoplasma polymerase chain reaction protocol. RESULTS: Animals were clinically healthy and not infested by ectoparasites. Although housekeeping gene DNA was successfully amplified, all the Barbary sheep samples tested negative for Mycoplasma sp. CONCLUSION: Notwithstanding the negative results, molecular pathogen surveys on Barbary sheep and other exotic wild mammals may provide insights regarding infection of endangered species caused by captivity stress in association with exposure to new pathogens worldwide.

Rapid, actionable diagnosis of urban epidemic leptospirosis using a pathogenic Leptospira lipL32-based real-time PCR assay.

BACKGROUND: With a conservatively estimated 1 million cases of leptospirosis worldwide and a 5-10% fatality rate, the rapid diagnosis of leptospirosis leading to effective clinical and public health decision making is of high importance, and yet remains a challenge. METHODOLOGY: Based on parallel, population-based studies in two leptospirosis-endemic regions in Brazil, a real-time PCR assay which detects lipL32, a gene specifically present in pathogenic Leptospira, was assessed for the diagnostic effectiveness and accuracy. Patients identified by active hospital-based surveillance in Salvador and Curitiba during large urban leptospirosis epidemics were tested. Real-time PCR reactions were performed with DNA-extracted samples obtained from 127 confirmed and 23 unconfirmed cases suspected of leptospirosis, 122 patients with an acute febrile illness other than leptospirosis, and 60 healthy blood donors. PRINCIPAL FINDINGS: The PCR assay had a limit of detection of 280 Leptospira genomic equivalents/mL. Sensitivity for confirmed cases was 61% for whole blood and 29% for serum samples. Sensitivity was higher (86%) for samples collected within the first 6 days after onset of illness compared to those collected after 7 days (34%). The real-time PCR assay was able to detect leptospiral DNA in blood from 56% of serological non-confirmed cases. The overall specificity of the assay was 99%. CONCLUSIONS: These findings indicate that real-time PCR may be a reliable tool for early diagnosis of leptospirosis, which is decisive for clinical management of severe and life-threatening cases and for public health decision making.

Microscopic and molecular identification of hemotropic mycoplasmas in South American coatis (Nasua nasua).

Hemoplasmas were detected in two apparently healthy captive South American coatis (Nasua nasua) from southern Brazil during an investigation for vector-borne pathogens. Blood was subjected to packed cell volume (PCV) determination, a commercial real-time PCR panel for the detection of Anaplasma spp., Babesia spp., Bartonella spp., Hepatozoon spp., Leishmania spp., Mycoplasma haemofelis, 'Candidatus Mycoplasma turicensis', 'Candidatus Mycoplasma haemominutum', Neorickettsia risticii, Rickettsia rickettsii and Leptospira spp., and a pan-hemoplasma conventional PCR assay. PCV was normal, but both coatis tested positive for hemoplasmas and negative for all the remaining pathogens tested. Using different techniques for microscopy (light, confocal or SEM), structures compatible with hemoplasmas were identified. Sequencing of the 16S rRNA gene identified an organism resembling Mycoplasma haemofelis and another hemotropic Mycoplasma sp., with a sequence identity of 96.8% to a Mycoplasma sp. previously detected in capybaras.

Occurrence of hemotropic mycoplasmas in non-human primates (Alouatta caraya, Sapajus nigritus and Callithrix jacchus) of southern Brazil.

Hemoplasmas, the erythrocyte-associated mycoplasmas, have been detected in several primates, causing mostly subclinical infection. This study aimed to determine the prevalence of hemoplasma infection in captive and free-ranging monkeys from southern Brazil, as well as factors and hematological abnormalities associated with infection. Blood samples from 40 non-human primates (NHP) were tested for hemoplasmas and coinfections. An overall of 10/40 (25.0%) NHP tested positive for hemoplasmas using PCR-based assays, including 9/14 (64.3%) black howler monkeys (Alouatta caraya) and 1/24 (4.2%) black-horned capuchin (Sapajus nigritus). Infection was not statistically associated with anemia, but wild-born monkeys and male black howler monkeys were more likely to be positive when compared with captive-born animals and female black howler monkeys, respectively. The sequences from the black howler monkey hemoplasma were similar (94% identity) to the squirrel monkey hemoplasma ("Candidatus Mycoplasma kahanei") and were phylogenetically located in a different cluster when compared to the human hemoplasma ("Candidatus Mycoplasma haemohominis").

Ehrlichia sp. infection in carthorses of low-income owners, Southern Brazil.

Although well established in dogs, Ehrlichia sp. infection has been scarcely reported in horses. The aim was to perform a comprehensive serological and molecular survey for the detection of Ehrlichia spp. in carthorses from Southern Brazil. Blood samples from 190 carthorses from Paraná State were sampled. Horses were also tested for Borrelia burgdorferi and Anaplasma phagocytophilum. Anti-Ehrlichia sp. antibodies were detected by a commercial rapid ELISA, and immunofluorescence antibody assays (IFA) with E. chaffeensis and E. canis as crude antigens. The molecular and phylogenetic analysis of Ehrlichia sp. was based on 16S rRNA and dsb genes. A total of 52 (27.4%), 4 (2.1%), and 3 (1.6%) horses were positive for Ehrlichia spp., Anaplasma spp. and Borrelia burgdorferi, respectively, by the commercial rapid ELISA. Thirty-eight (20.0%) and 37 (19.5%) horses showed anti-E. chaffeensis and anti-E. canis antibodies by IFA, respectively. One blood sample that also showed anti-E. chaffeensis antibodies was PCR positive for the 16S rRNA and dsb genes of Ehrlichia spp., showing an identity of>98.0% to the uncultured Ehrlichia sp. previously detected in Brazilian jaguars (Panthera onca). Anti-Ehrlichia sp. antibodies and Ehrlichia DNA were detected in carthorses from Southern Brazil, which may post public health concerns due to intimate contact with low-income owners. This is the first report of a natural infection of this bacteria in horses from South America. Clinical signs and the tick vector remain unknown.

Serological and molecular detection of Theileria equi in sport horses of northeastern Brazil.

Theileriosis is a worldwide protozoal tick-borne disease caused by Theileria equi, which may produce a variety of clinical signs and turn infected horses into lifetime carriers. This study has aimed to perform a serological and molecular detection of T. equi and associated factors in sports horses from six areas of northeastern Brazil. In overall, 59.6% horses were positive by indirect immunofluorescence assay and 50.4% by polymerase chain reaction. No significant association was found when presence of ticks, age, gender, anemia or total plasma proteins was analyzed with seropositivity and molecular techniques. Although a significant association of infection was found in two cities. Thus, local risk factors other than presence of ticks, horse age, gender, anemia and total plasmatic proteins may dictate prevalence of T. equi infection in sports horses, even in highly endemic areas with no control of infection prior to horse competitions.

An Optimized Method for Quantification of Pathogenic Leptospira in Environmental Water Samples.

Leptospirosis is a zoonotic disease usually acquired by contact with water contaminated with urine of infected animals. However, few molecular methods have been used to monitor or quantify pathogenic Leptospira in environmental water samples. Here we optimized a DNA extraction method for the quantification of leptospires using a previously described Taqman-based qPCR method targeting lipL32, a gene unique to and highly conserved in pathogenic Leptospira. QIAamp DNA mini, MO BIO PowerWater DNA and PowerSoil DNA Isolation kits were evaluated to extract DNA from sewage, pond, river and ultrapure water samples spiked with leptospires. Performance of each kit varied with sample type. Sample processing methods were further evaluated and optimized using the PowerSoil DNA kit due to its performance on turbid water samples and reproducibility. Centrifugation speeds, water volumes and use of Escherichia coli as a carrier were compared to improve DNA recovery. All matrices showed a strong linearity in a range of concentrations from 106 to 10° leptospires/mL and lower limits of detection ranging from <1 cell /ml for river water to 36 cells/mL for ultrapure water with E. coli as a carrier. In conclusion, we optimized a method to quantify pathogenic Leptospira in environmental waters (river, pond and sewage) which consists of the concentration of 40 mL samples by centrifugation at 15,000×g for 20 minutes at 4°C, followed by DNA extraction with the PowerSoil DNA Isolation kit. Although the method described herein needs to be validated in environmental studies, it potentially provides the opportunity for effective, timely and sensitive assessment of environmental leptospiral burden.

Detection of Ehrlichia canis, Babesia vogeli, and Toxoplasma gondii DNA in the Brain of Dogs Naturally Infected with Leishmania infantum.

The aims of this study were to investigate the presence of Leishmania infantum and possible co-infection with Anaplasma platys , Babesia vogeli, Ehrlichia canis , and Toxoplasma gondii in the brain of 24 dogs naturally infected by L. infantum . A total of 24 mongrel adult dogs (22 clinically affected, 2 with neurological signs, and 2 subclinically infected) aged between 2 and 5 yr, naturally infected by visceral leishmaniasis, were selected. Fragments from meninges, frontal cortex, thalamus, cerebellum, and choroid plexus of the lateral ventricles and fourth ventricle were collected, mixed, and tested by real-time polymerase chain reaction (qPCR). Leishmania infantum DNA was detected in 95.8% (23/24) of the infected dogs, including the subclinically infected. A total of 14/24 (58.3%) dogs were co-infected by E. canis and L. infantum , 4/24 (16.7%) were co-infected by E. canis , B. vogeli, and L. infantum , 2/24 (8.3%) were co-infected by B. vogeli and L. infantum , and 1/24 (4.2%) dog was co-infected by E. canis , B. vogeli, T. gondii , and L. infantum . All 24 brain samples tested negative for A. platys . These results demonstrate that L. infantum is able to penetrate into the brain parenchyma, either alone or in association to other zoonotic pathogens. In addition, qPCR could be considered for adequate evaluation of Leishmania in the brain tissue of dogs with neurological signs that have died.

Seroepidemiological survey of Theileria equi and Babesia caballi in horses from a rural and from urban areas of Paraná State, southern Brazil.

The objective of this study was to evaluate the seroepidemiological data of Babesia caballi and Theileria equi in horses from a rural settlement and carthorses from urban areas of Paraná State, southern Brazil. A total of 198 horses, including 32 from the rural settlement and 166 carthorses from Colombo (n=48), Pinhais (n=76), Londrina (n=24), and Curitiba city (n=18) was sampled and tested using a commercial competitive inhibition ELISA (cELISA) test. Out of the 198 horses, 193 (97.5%) were seropositive for at least one piroplasm species. Antibodies to T. equi were detected in 155/198 horses (78.3%), antibodies to B. caballi were detected in 137/198 horses (69.2%), and antibodies to both were detected in 99/198 (50.0%) horses. Horses living in the rural settlement and Colombo were more likely to be seropositive to T. equi than those in Curitiba (p<0.05). Horses older than 5 years were more likely to be seropositive for T. equi than those younger than 5 years (p<0.05). No significant association was found between gender or the presence of ticks and seropositivity to T. equi (p>0.05). In conclusion, the high seroprevalences to B. caballi and T. equi observed in this study emphasize that active surveillance programs are critical for monitoring animal health status, particularly because carthorses may act as urban disseminators of these piroplasms.

Longitudinal analysis of serological tests officially adopted by the Brazilian Ministry of Health for the diagnosis of canine visceral leishmaniasis in dogs vaccinated with Leishmune®.

Development of vaccines against canine visceral leishmaniasis (CVL) may provide a prophylactic barrier, but antibody response detected by standard diagnostic techniques may not separate vaccinated from naturally infected dogs. Moreover, anti-Leishmania antibody levels in vaccinated dogs may be detectable for months. Accordingly, the aim of the present study was to comparatively evaluate an "in-house" ELISA with three serological tests officially adopted by the Brazilian Ministry of Health for the diagnosis of CVL in dogs vaccinated with Leishmune(®). A total of 18 mongrel dogs were submitted to a complete protocol of the vaccine, monitored and evaluated in 5 times (T0-T4) up to 180 days after T0. Twenty-one days after the first dose (T1), 50% of the dogs were seropositive by the "in-house" ELISA and 5.5% by IFAT, while by the official ELISA and DPP(®) CVL rapid test all dogs tested negative. At time T2, 42 days after of the first dose, 100%, 83.3%, 11.1%, and 5.5% of the dogs were seropositive by the "in-house" ELISA, IFAT, official ELISA kit and the DPP(®) CVL rapid test, respectively. Ninety days after the first dose (T3), 100%, 83.3%, 72.2% and 33.3% of the dogs were seropositive by the "in-house" ELISA, official ELISA kit, IFAT, and the DPP(®) CVL rapid test, respectively. Finally, at time T4, 88.8%, 33.3%, 11.1% and 5.5% of the dogs were seropositive by the "in-house" ELISA, official ELISA kit, DPP(®) CVL rapid test and IFAT, respectively. In conclusion, dogs vaccinated with Leishmune(®) cross-react by an "in-house" ELISA and by the three official Brazilian serological tests for the diagnosis of canine visceral leishmaniasis up to six months after the first vaccine dose, and may be mistakenly diagnosed and removed.