Cristiane Silva Chitarra

A survey of tick-borne pathogens in dogs and their ticks in the Pantanal biome, Brazil.

Tick and blood samples collected from domestic dogs in the Brazilian Pantanal were tested by molecular methods for the presence of tick‐borne protozoa and bacteria. Among 320 sampled dogs, 3.13% were infected by Babesia vogeli (Piroplasmida: Babesiidae), 8.75% by Hepatozoon canis (Eucoccidiorida: Hepatozoidae), 7.19% by Anaplasma platys (Rickettsiales: Anaplasmataceae), and 0.94% by an unclassified Anaplasma sp. In three tick species collected from dogs, the following tick‐borne agents were detected: (a) B. vogeli, An. platys and Ehrlichia canis (Rickettsiales: Anaplasmataceae), infecting Rhipicephalus sanguineus sensu lato (Ixodida: Ixodidae) ticks; (b) H. canis, an unclassified Anaplasma sp. and Rickettsia amblyommii (Rickettsiales: Rickettsiaceae), infecting Amblyomma cajennense sensu lato (Ixodida: Ixodidae) ticks, and (c) Rickettsia sp. strain Atlantic rainforest, an emerging human pathogen, infecting Amblyomma ovale ticks. Molecular analysis, based on a mitochondrial gene, revealed that the Am. cajennense s.l. ticks of the present study corresponded to Amblyomma sculptum, a member of the Am. cajennense species complex, and that Rh. sanguineus s.l. belonged to the tropical lineage. Whereas dogs are exposed to a number of tick‐borne bacterial and protozoan agents in the Pantanal biome, humans are potentially exposed to infection by spotted fever group rickettsiae (e.g. R. amblyommii and Rickettsia sp. strain Atlantic rainforest) because both Am. sculptum and Am. ovale are among the most important human‐biting ticks in Brazil.

Rickettsia parkeri infecting free-living Amblyomma triste ticks in the Brazilian Pantanal.

The present study evaluated the infection of rickettsiae in 151 Rhipicephalus sanguineus, 59 Amblyomma ovale, 166 Amblyomma triste, one Amblyomma dissimile and four Amblyomma dubitatum ticks collected in the municipality of Poconé, State of Mato Grosso, within the Pantanal biome of Brazil. Ticks were individually processed by the hemolymph test with Gimenez staining, isolation of rickettsia in Vero cell culture by the shell vial technique, and polymerase chain reaction (PCR) targeting the citrate synthase rickettsial gene. Through the shell vial technique, rickettsiae were successfully isolated and established in Vero cell culture from one free-living A. triste female tick, which previously showed to contain Rickettsia-like organisms by the hemolymph test. Molecular characterization of the rickettsial isolate was achieved through DNA partial sequences of three rickettsial genes (gltA, ompA, ompB), which showed to be all 100% identical to Rickettsia parkeri. After testing all ticks by PCR, the frequency of R. parkeri infection was 7.23% (12/166) in A. triste adult ticks. The remaining ticks were negative by PCR. This is the first report of in vitro isolation of R. parkeri in the Pantanal biome, confirming the occurrence of this emerging rickettsial pathogen in this natural area of South America.

Minimum infection rate of Ehrlichia minasensis in Rhipicephalus microplus and Amblyomma sculptum ticks in Brazil.

A new genotype phylogenetically close to Ehrlichia canis named as Ehrlichia minasensis was identified infecting cattle and deer in Canada, as well as Rhipicephalus microplus ticks and cattle in Brazil. Although it was detected in R. microplus, little is known about the epidemiology of this ehrlichiosis, especially in other tick species. This study evaluated the minimum infection rate of E. minasensis in Amblyomma sculptum and R. microplus ticks from locations where naturally infected cattle were previously detected. Overall, 45 engorged R. microplus ticks after molting [43 pools of adults (13.4%), and 2 pools of nymphs (4%)], and 42 engorged females post-oviposition (30.6%) (p=0.008) were positive by PCR for Ehrlichia sp. using the dsb, 16S rRNA and TRP36 genes, making a total of 87 R. microplus samples positive for Ehrlichia spp. (17.1%, IC 95% 14.01-20.75%). The partial sequences generated in the present study were 99-100% similar to the dsb DNA sequence of E. minasensis genotypes UFMG-EV and UFMT-BV, respectively, 100% similar to the 16S rRNA sequence of the E. minasensis genotype BOV2010 from Canada, and 99% similar to the TRP36 sequence of the Ehrlichia sp. UFMT-BV. The results of this study confirm the occurrence of transstadial transmission of this agent in R. microplus ticks and highlight the importance of R. microplus in the epidemiology and transmission of ehrlichiosis in cattle. No A. sculptum ticks were positive by PCR for E. minasensis.

Molecular detection of Bartonella clarridgeiae in domestic cats from Midwest Brazil

Cat scratch disease (CSD) is caused by Bartonella henselae a hemotropic bacterium that infects human, domestic and wild mammals. Closely related to B. henselae, the B. clarridgeiae comprises approximately 10–30% of Bartonella isolates from clinically healthy cats and was found in cardiac and hepatic lesions in dogs.1 Moreover, B. clarridgeiae has been serologically associated with CSD-like illness in humans.2 Cats are considered the major reservoir and carriers of B. henselae, B. clarridgeiae and B. koehlerae. Bartonella spp. can be a vector transmitted by flea to susceptible cats. Few cats naturally infected have clinical signs. Medical problems of cats associated with Bartonella infections are variable, ranging from transient fever and lymphadenopathy to abscesses or microabscesses in different organs, endocarditis, and central nervous signs.1 Transmission from cats to humans and dogs mainly occurs through cat scratches or bites. In humans, this infection may be asymptomatic and can disappear spontaneously without any treatment; however, in some cases the disease may be fatal if left untreated.3 In order to identify arthropod-borne pathogens in domestic feline, 182 cats from different places of Cuiabá and Varzea Grande cities, both located in Mato Grosso State, Midwest Brazil, have been analyzed. Cats were seen at the Veterinary Hospital of the Federal University of Mato Grosso (HOVET-UFMT), Veterinary Hospital of the University of Cuiabá (UNIC-HV), and animal shelters and Zoonosis Control Centers (ZCC) of Cuiabá and Várzea Grande cities. Sample collection was conducted in agreement with the Ethical Principles for Animal Research under the institutional watch of the UFMT Committee for Ethics in Animal Research. Collected blood samples were subjected to a DNA extraction using the Axyprep Blood Genomic DNA Miniprep Kit (Axygen Biosciences, Hangzhou, China). Extracted DNA was then used as a template for Bartonella spp. PCR assay using the primer BARTON-1 (5-TAACCGATATTGGTTGTGTTGAAG3) and BARTON-2 (5-TAAAGCTAGAAAGTCTGGCAACATAACG3), which amplifies a segment of 585–588 bp of Riboflavin synthase C (ribC) gene of Bartonella genus.4 Three (1.6%) cats were positive to the presence of Bartonella DNA according to PCR targeting portions of the ribC gene. Partial DNA sequences of all PCR-positive samples were generated yielding a sequence (GenBank accession: KR092386) that was identical to KC331014 and HM588660 and 99% similarity to HQ012585, KC331017, KC331014, which corresponds to B. clarridgeiae sequences available in GenBank. The positive cats were found in the ZCC of Várzea Grande city. Usually, these animals are taken off the streets and kept until adoption. Boulouis et al. observed that stray cats present higher prevalence of Bartonella spp. infection than pet cats, mainly due to close contact between infected animals in large groups of cats, demonstrating that differences in the occurrence of the infection are associated with the type of feline population studied.5 Due to vigorous flea control among cat populations, the prevalence of Bartonella infection in cats have reduced and the risk of Bartonella-associated disease in pet owners decreased. However, cat owners and animal health professionals should be cautioned to avoid behaviors that increase the risk of animal bites or scratches, since the feline population acts as a source of zoonotic agents and represents a potential risk of infection.1 In Brazil, the occurrence of Bartonella infection in human was previously described. In the State of Rio de Janeiro, 41.6% of clinically asymptomatic HIV-positive patients were found seropositive for Bartonella species.3 In fact, veterinarians and medical doctors should consider the presence of these zoonotic pathogens in their diagnostic routine.

Prevalência de Streptococcus suis tipo 2 por meio da técnica de reação em cadeia da polimerase em suínos abatidos no Estado do Mato Grosso

Streptococcus suis is a pathogen that affects the industrial production of swine worldwide. It is extremely important, because it is associated with pigs and humans diseases. The aim of this study was to determine the prevalence of Streptococcus suis type 2 in 201 samples of tonsils from clinically healthy animals by the PCR technique. The samples positive for S. suis type 2 were tested for the gene encoding extracellular factors (ef). The results showed that the prevalence (23.38%) was higher than other recent survey in the State, demonstrating that the PCR is a more sensitive method in relation to the bacterial isolation. There was a low occurrence of ef* gene in samples (1.49%) showing great importance to local swine population, because negative strains are potentially less virulent that positive strains.

Molecular detection of tick-borne protozoan parasites in a population of domestic cats in midwestern Brazil.

Some tick-borne pathogens that infect domestic cats have been considered emergent in veterinary medicine. Occurrences of Hepatozoon spp., Babesia spp. and Cytauxzoon spp. have been described in several regions of Brazil. This paper offers a comprehensive analysis of the 18S rRNA gene of a Hepatozoon sp. strain detected in domestic cats in the metropolitan area of Cuiabá, in Midwestern Brazil. Based on a molecular analysis, we detected the presence of Hepatozoon species circulating among cats in this region. The aforementioned strain is closely related to other isolates of H. felis detected in wild felids. Moreover, a phylogenetic analysis indicates that this genotype is grouped into a clade of 18S rRNA sequences previously described for the genus Hepatozoon in wild felids around the world. Hepatozoon felis strains detected in cats from Spain and Israel showed, respectively, 98% and 97% identity to our sequence and are clustered on a separate branch of the phylogenetic tree. This finding suggests a high diversity of Hepatozoon genotypes occurring in cats in Europe and South America. None of the analyzed cats were positive for Babesia spp. or Cytauxzoon spp. by PCR analysis.

Padronização da técnica de nanopartícula de ouro não modificada (AuNPs) para detecção de Actinobacillus pleuropneumoniae em pulmões de suínos

Based on diagnostic tests for the detection of nucleic acids without amplification through the use of gold nanoparticles (AuNPs) have been described for various diseases. This study aimed to develop a technique of unmodified AuNPs to detect Actinobacillus pleuropneumoniae (App). We used 70 lung samples from pigs, 17 with and 53 without characteristic lesions of pneumonia, to detect App. The primer used was based on ApxIV gene. The AuNPs test had a sensitivity of 93.8% and specificity of 84.6% when compared with PCR detection. The results showed good agreement between AuNPs and PCR testing, and the technique can be used as an alternative to conventional tests, since it is quick and easy, and does not require implementation infrastructure and skilled labor.

Identification of Pasteurella multocida transcribed genes in porcine lungs through RNAseq

Pasteurella multocida is one of the most important pathogen that causes pneumonia in swine. Although several virulence factors are known, the pathogenesis of this bacterium is not well-studied. Therefore, to study the pathogenesis of P. multocida infection in porcine lung, next-generation RNA sequencing was used to compare the transcriptomes of P. multocida grown in vivo and in vitro, respectively. After P. multocida infection a total of 704 genes were expressed in vitro, 1422 genes were expressed in vivo, and 237 genes were differentially expressed based on statistical analyses, padj of ≤0.1. Genes encoding ribosomal proteins or other products that function in the regulation of transcription and translation were downregulated, whereas genes whose products affected cellular processes (protein transport and RNA degradation) and metabolic pathways, such as those of amino acid metabolism and nucleotide metabolism, were upregulated in vitro compared with in vivo. This study shows that differentially expressed genes in P. multocida regulate pathways that operate during stress, iron capture, heat shock, and nitrogen regulation. However, extensive investigation of the pathogenic mechanism of P. multocida is still required.

Nephrin gene expression in chronic kidney disease of dogs with Leishmania (Leishmania) infantum chagasi

Visceral leishmaniasis caused by Leishmania (Leishmania) infantum chagasi is the most severe form of the disease. In Brazil, this disease is transmitted through the bite of sand flies of the genus Lutzomyia.1 The clinical signs of the disease in canines, which are similar to those in humans, depend on the individual’s humoral and cellular response, and kidney disease is the main cause of death.2 In visceral leishmaniasis nephropathies caused by glomerular diseases are a common cause of chronic renal failure.2–4 Glomerular podocyte injury causes proteinuria and results in tubular and interstitial injury.5 Thus, the aim of the study was to quantify nephrin gene expression in dogs suffering from chronic kidney disease (CKD) associated with visceral leishmaniasis. Sixty-nine dogs with CKD were diagnosed based on their history, the presence of azotemia and isosthenuria, and were grouped according to classification proposed by the International Renal Interest Society (IRIS). Urine, blood and bone marrow samples were collected by jugular venipuncture, cystocentesis and/or urethral catheter, and sternal puncture, respectively. This study was approved by and conducted according to the guidelines of the Ethics Committee on Animal Use of the Federal University of Mato Grosso (Protocol # 23108.043331/129). The DNA extracted from blood and bone marrow was subjected to PCR detection of L. (L.) infantum chagasi. Cell pellets were obtained by centrifuging 10 mL of urine for RNA extraction, followed by RT-qPCR to quantify nephrin gene expression. Supernatant from centrifuged urine was used for biochemical determination of proteinuria. Nephrin was quantified in the urinary sediment of 47 dogs and L. (L.) infantum chagasi was detected in the bone marrow and/or blood of 11 dogs. The relative quantitation of nephrin was lower in dogs in advanced stages of renal disease than in the initial stages, and those positive for L. L. infantum chagasi showed no correlation with the presence of the parasite (r = −0.35). The quantitation of nephrin expression according to the stage of CKD and its association with L. L. infantum chagasi have not been reported previously. Kidney disease is an important condition in the clinical assessment of small animals, in view of the high prevalence and severity of its subsequent clinical manifestations. Moreover, owing to the zoonotic potential of visceral leishmaniasis associated with the presence of asymptomaticity in dogs, the early detection of kidney disease enables the evolution of the disease to be monitored. Visceral leishmaniasis impairs the immune system and interferes with the inflammatory response, resulting in several changes that culminate in death from renal failure. Knowing that canine cases precede the occurrence of the disease in humans, it is important to identify the changes that may occur in both species. Therefore, dogs also serve as a study model of this disease, assisting in the establishment of a more accurate prognosis for humans, and is a control measure that requires fast and accurate diagnostic tools. Hence, the quantitation of nephrin gene expression in urinary sediment is a practical and non-invasive tool for monitoring the development of kidney disease.