Molecular characterization of Hepatozoon spp. infection in endangered Indian wild felids and canids

Hepatozoon species are parasites that infect a wide variety of domestic and wild animals. The objective of this study was to perform the molecular detection and characterization of Hepatozoon spp. in Asiatic lion, Indian tiger, Indian leopard, Indian wild dog, Indian domestic dog and cat based on partial 18S rRNA gene sequences from Hepatozoon spp. in the naturally infected animals. Hepatozoon spp. could be detected in blood samples of 5 out of 9 Asiatic lions, 2 out of 5 Indian tigers, 2 out of 4 Indian leopards and 2 out of 2 Indian wild dogs and, 2 out of 4 domestic cats and 2 out of 3 domestic dog samples by PCR. Sequencing of PCR amplicon and BLAST analysis of partial 18S rRNA gene sequences indicated that the Hepatozoon spp. in Asiatic lion, Bengal tiger, Indian leopard and domestic cat was Hepatozoon felis (98-99% similarity) and in the Indian wild and domestic dog the phylogenetic neighbour was Hepatozoon canis (97-100% similarity). Presence of H. felis and H. canis in both domestic and wild animals suggested that they are not host specific and the same parasite causes infection in domestic and wild felids and canids in India and from different parts of the world. To our knowledge, this is the first report on detection and molecular characterization of H. felis infection in Asiatic lions, Indian tigers, Indian leopards and H. canis in Indian wild dog. Hepatozoon spp. may be a potential pathogen and an opportunistic parasite in immuno-compromised animals and could thus represent a threat to endangered Indian wild felids and canids.     

Molecular characterization of feline Hepatozoon species from Brazil

Feline Hepatozoon species from Brazil was molecular identified and characterized for the first time in S?o Paulo state, Brazil. Partial sequences of the 18S rRNA gene from the Hepatozoon from three naturally infected cats were analyzed. Sequences revealed that feline Hepatozoon was closely related to the canine Hepatozoon canis from Brazil.     

Diagnosis of Hepatozoon spp. in Amblyomma ovale and its experimental transmission in domestic dogs in Brazil

Transmission of Hepatozoon spp. to dogs was investigated using four species of ixodid ticks: Rhipicephalus sanguineus, Amblyomma aureolatum, Amblyomma ovale and Amblyomma cajennense. We collected completely or partially engorged adult ticks of these species from dogs that were naturally infested and positive for Hepatozoon spp. We selected some of these ixodids and inoculated them orally in four negative dogs. The other ticks were dissected and examined for oocysts. Of all dogs inoculated orally with R. sanguineus, A. aureolatum, A. cajennense and A. ovale, only the animal that received the macerate of A. ovale was positive; evidence (gametocytes in peripheral blood) of infection was found 63 days after inoculation. Among all dissected ticks, we found only two oocysts; these were similar to those of Hepatozoon canis, and both were recovered from a single A. ovale specimen. We inoculated sporozoites recovered from the oocysts intraperitoneally into a Hepatozoon spp. negative dog, and circulating gametocytes were detected 84 days later. Our study demonstrated that A. ovale can be a vector of Hepatozoon spp. in Brazil.     

Molecular detection of Babesia rossi and Hepatozoon sp. in African wild dogs (Lycaon pictus) in South Africa

Blood specimens from wild dogs (n=301) were obtained from De Wildt Cheetah and Wildlife Centre (Pretoria) and five game reserves (4 in the North-West Province and 1 in Limpopo Province), South Africa. Specimens were screened for Babesia, Theileria, Hepatozoon and Ehrlichia/Anaplasma species using PCR and Reverse Line Blot (RLB) assays. Positive results were obtained in 18 (6%) wild dogs. Sixteen specimens were found positive for Babesia rossi and two dogs were Hepatozoon sp. positive. It appears that these tick-borne pathogens are not widely distributed in wild dog populations.     

Phylogenetic and host-parasite relationship analysis of Henneguya multiplasmodialis n. sp. infecting Pseudoplatystoma spp. in Brazilian Pantanal wetland

A new species of the genus Henneguya (Henneguya multiplasmodialis n. sp.) was found infecting the gills of three of 89 specimens (3.3%) of Pseudoplatystoma corruscans and two of 79 specimens (2.6%) of Pseudoplatystoma reticulatum from rivers in the Pantanal wetland, Brazil. Partial sequencing of the 18S rDNA gene of the spores obtained from one plasmodium from the gills of P. corruscans and other one from the gills of P. reticulatum, respectively, resulted in a total of 1560 and 1147 base pairs. As the spores of H. multiplasmodialis n. sp. resemble those of Henneguya corruscans, which is also a parasite of P. corruscans, sequencing of the 18S rDNA gene of the spores of H. corruscans found on P. corruscans caught in the Brazilian Pantanal wetland was also provided to avoid any taxonomic pendency between these two species, resulting in 1913 base pairs. The sequences of H. multiplasmodialis n. sp. parasite of P. corruscans and P. reticulatum and H. corruscans did not match any of the Myxozoa available in the GenBank. The similarity of H. multiplasmodialis n. sp. obtained from P. corruscans to that from P. reticulatum was of 99.7%. Phylogeny revealed a strong tendency among Henneguya species to form clades based on the order and/or family of the host fish. H. multiplasmodialis n. sp. clustered in a clade with Henneguya eirasi and H. corruscans, which are also parasites of siluriforms of the family Pimelodidae and, together with the clade composed of Henneguya spp. parasites of siluriforms of the family Ictaluridae, formed a monophyletic clade of parasites of siluriform hosts. The histological study revealed that the wall of the plasmodia of H. multiplasmodialis n. sp. were covered with a stratified epithelium rich in club cells and supported by a layer of connective tissue. The interior of the plasmodia had a network of septa that divided the plasmodia into numerous compartments. The septa were composed of connective tissue also covered on both sides with a stratified epithelium rich in club cells. Inflammatory infiltrate was found in the tissue surrounding the plasmodia as well as in the septa.     

Molecular genetic comparison of Onchocerca sp. infecting dogs in Europe with other spirurid nematodes including Onchocerca lienalis

In the past 15 years, subconjunctival onchocercosis has been reported from 63 dogs in south-western United States (Arizona, California, Utah) and Southern and Central Europe (Germany, Greece, Hungary, Portugal, Switzerland). To reveal the taxonomic status of the parasite responsible for these infections, fragments of the mitochondrial cytochrome oxidase subunit I (COI) and NADH dehydrogenase subunit 5 (ND5) genes of three European strains of canine Onchocerca sp. and the 16S ribosomal RNA (16S rRNA) gene of their Wolbachia endosymbionts were sequenced and compared to the homologous sequences of other spirurid nematodes. The evolutionary divergence between COI and ND5 gene sequences of Greek, Hungarian and Portuguese strains of canine Onchocerca sp. were similar in magnitude to that seen within Thelazia callipaeda or Onchocerca lienalis. The evolutionary divergence between the sequences of canine Onchocerca sp. and other Onchocerca spp. including O. lienalis were similar or higher in magnitude to that seen between other Onchocerca spp. The results of the current and earlier phylogenetic analyses indicate that canine Onchocerca sp. separated from other Onchocerca spp. early in the evolution. Based on the similar clinical pictures, the identical morphology of nematodes and the sequence analyses of COI and ND5 genes of the worms and 16S rRNA gene of their wolbachiae, the Onchocerca worms isolated from European dogs appear to belong to the same species. The results support the earlier biological and morphological arguments that a distinct species, most likely O. lupi originally described from the subconjunctival tissues of a Caucasian wolf is responsible for canine ocular onchocercosis in Europe.     

Molecular and phylogenetic characterization of Theileria spp. parasites in autochthonous bovines (Mirandesa breed) in Portugal

A survey was conducted during the months of April-June 2003 in the northeast Portugal (Bragan?a district) in order to characterize the hemoparasite population of an autochthonous Mirandesa breed of Bos taurus. The polymerase chain reaction (PCR) analysis of the bovine blood revealed that 3 out of 116 animals were infected with Theileria and/or Babesia parasites, while reverse line blot hybridisation (RLB) analysis showed that these animals were infected with Theileria buffeli/orientalis. Cloning and sequencing confirmed the RLB results. Database sequence searches combined with phylogenetic analysis of the partial 18S ribosomal RNA gene sequences obtained enabled us to place the parasites in question as members of the T. buffeli/orientalis group, confirming the PCR/RLB diagnosis.     

Genotypic characterization and phylogenetic analysis of Cryptosporidium sp. from domestic animals in Brazil

The purpose of the present study was the genetic characterization, sequencing and phylogenetic analysis of 18S rDNA sequences of Cryptosporidium isolates obtained from different animal hosts in Brazil. Fecal samples containing Cryptosporidium oocysts were obtained from chickens, ducks, quails, guinea pigs, dairy calves, dogs and cats. For amplification of 18S rDNA sequences the Secondary-PCR product of the extracted DNA from fecal suspension of each studied animal was utilized. The primary genetic characterization of Cryptosporidium sp. was performed using RFLP with the enzymes SspI and VspI. DNA samples were sequenced and subjected to phylogenetic analysis. The results showed C. baileyi infecting two ducks and one quail and C. melagridis infecting one chicken. The sequences obtained from Cryptosporidium sp. infecting guinea pigs were not identified within groups of known Cryptosporidium species. The isolates found parasitizing cats and one dog were diagnosed as C. felis and C. canis, respectively. One isolate of calf origin was identified as C. parvum. The phylogenetic analysis showed clear distribution of isolates between two Cryptosporidium sp. groups according to their gastric or intestinal parasitism. A great genetic distance was observed between C. felis and C. canis from Brazil when compared to the reference sequences obtained from GenBank. The results obtained during this study constitute the first report of rDNA sequences from C. baileyi, C. meleagridis, C. felis, C. canis and C. parvum isolated in Brazil.     

Occurrence and molecular characterization of Bartonella spp. and hemoplasmas in neotropical primates from Brazilian Amazon

Little is known about the prevalence and genetic diversity of Bartonella spp. and hemoplasmas in nonhuman primates (NHP). The present study aimed to investigate the occurrence of and assess the phylogenetic position of Bartonella spp. and hemoplasma species infecting neotropical NHP from Brazilian Amazon. From 2009 to 2013, a total of 98 blood samples from NHP belonging to the Family Cebidae were collected in the island of São Luís, state of Maranhão, of which 87 NHP were from Wild Animal Screening Center (CETAS) in the municipality of São Luís, and 11 (9 Sapajus sp. and 2 Saimiri sciureus) were from NHP caught in the Sítio Aguahy Private Reserve. DNA samples were screened by previously described PCR protocols for amplifying Bartonella spp. and Mycoplasma spp. based on nuoG, gltA and 16S rRNA genes, respectively. Purified amplicons were submitted to sequencing and phylogenetic analysis. Bacteremia with one or more Bartonella spp. was not detected in NHP. Conversely, 35 NHP were PCR positive to Mycoplasma spp. The Blastn analysis of seven positive randomly selected sequencing products showed percentage of identity ranging from 95% to 99% with other primates hemoplasmas. The Maximum Likelihood phylogenetic analysis based on a 1510 bp of 16S rRNA gene showed the presence of two distinct clusters, positioned within Mycoplasma haemofelis and Mycoplasma suis groups. The phylogenetic assessment suggests the presence of a novel hemoplasma species in NHP from the Brazilian Amazon.     

Diagnosis of canine Hepatozoon spp. infection by quantitative PCR

Hepatozoon (H.) americanum and H. canis are the etiological agents of canine hepatozoonosis, a disease that is found worldwide and is also prevalent in the southeastern United States. Current laboratory diagnosis of canine hepatozoonosis caused by H. americanum is usually dependent on visual identification of Hepatozoon "onion skin cysts" in muscle biopsies, an approach that requires invasive sampling and can result in false negatives. We have developed a diagnostic method for detection of Hepatozoon spp. DNA that integrates nucleic acid extraction with extensive agitation to maximize DNA extraction efficiency. The DNA extracted from canine EDTA-whole blood is subjected to real-time PCR, and fluorescence resonance energy transfer (FRET) probes detect a signature polymorphism in the amplified DNA. This PCR method amplifies a fragment of the Hepatozoon 18S rDNA gene, detects as few as 7 genomic copies of Hepatozoon spp. per ml of blood with high specificity, and differentiates between H. americanum and H. canis amplicons. A surprising 300-fold increase of H. americanum 18S rDNA targets occurred during 3-0 days of storage of positive blood specimens. Examination of 614 EDTA-blood samples submitted mostly from the southeastern Unites States from dogs with suspected hepatozoonosis identified H. americanum in 167 samples (27.2%). An additional 14 samples (2.3%) were positive for H. canis, and 14 samples (2.3%) were positive for both H. americanum and H. canis. These results suggest that the Hepatozoon spp. 18S rDNA quantitative PCR may be a valuable tool that can improve diagnosis and therapy of canine hepatozoonosis.     

Molecular characterization of Hepatozoon species in a Leopard Cat (Prionailurus bengalensis) from Thailand

Abstract: Hepatozoon gamonts were observed by light microscopy in neutrophils of a male, wild‐caught Leopard Cat. Complete blood counts at presentation and 6 months later were unremarkable. Serologic tests were negative for both FIV and FeLV. A partial sequence of the 18S rRNA gene from the Hepatozoon found in the cat indicated that, compared with all species examined, the protozoan had the closest relationship (99.2% sequence similarity) with the Hepatozoon of the water python (Stegonotus cucullatus). The cat was clinically healthy at last report. Although Hepatozoon has been found in another wild cat in Thailand, this is the first report in a Leopard Cat. The pathogenicity of Hepatozoon in these cats remains uncertain.     

Molecular detection of Hepatozoon canis in dogs and ticks in Shaanxi province,China

Hepatozoon canis, transmitted by Rhipicephalus sanguineus, is a tick-borne pathogen and causes canine hepatozoonosis. Until now, only limited previous studies were conducted on the molecular detection and characterization of Hepatozoon sp. in dogs in China. Blood samples were collected from 93 sick dogs that were clinically diagnosed as babesiosis but tested negative for Babesia, and 103 apparently healthy dogs, as well as their infesting ticks in Xi’an and Hanzhong cities, Shaanxi province of China. PCR amplifying partial 18S rRNA gene was used to detect the DNA of Hepatozoon sp. Genetic and phylogenetic analysis were performed to determine the Hepatozoon species. Our results demonstrated that H. canis was identified from the sick dogs and the infested ticks in Hanzhong, with no significant differences of prevalence between both genders and ages. No positive blood or tick samples were found in Xi’an. Moreover, all the 18S rRNA gene sequences recovered from both dogs and the infested ticks showed a high genetic similarity with each other, and also presented a close relationship with other known sequences in and outside China. In conclusion, H. canis was identified in babesiosis-suspected dogs and ticks infesting them in Shaanxi, China, although the association between clinical signs and H. canis need further study.     

Molecular survey of Rickettsia spp. in sick dogs in Italy

The aim of this retrospective study was to investigate the prevalence of Rickettsia spp. DNA in the blood of sick dogs from Italy. Canine blood samples (n=650) submitted for molecular testing of Rickettsia spp. to a diagnostic laboratory from February 2003 to March 2006 were studied. The Rickettsia spp. DNA detection was performed by Light Cycler real-time PCR using hybridization probes separately conducted with specific primers and probes. The total percentage of Rickettsia spp.-positive dog samples was 1.5% (10 out of 650). The percentage of Rickettsia spp.-positive dog samples submitted from north, central and southern Italy were 0.4% (1/248), 1.4% (3/219) and 3.3% (6/183), respectively. Five out of 138 dogs (3.6%) from Sicily were positive on Rickettsia PCR testing. A statistical difference was found between the percentages of positive samples from the Yorkshire terrier group (10.7%) compared with the mixed breed group (0.7%). No statistical differences were found between seasonal period, region and gender. Based on molecular data, there is infrequent rickettsiemia in dogs.     

Occurrence and characterization of gastric Helicobacter spp. in naturally infected dogs

Helicobacter-like organisms are frequently observed in the stomach of dogs but the relationship between these microorganisms and gastric pathology has not been clearly established. Different species of helicobacters are known to be present in the canine stomach but their specific prevalence in naturally infected dogs is unknown. The aims of this study were to isolate and characterize helicobacters in canine gastric biopsies, to compare the commonly used tests for the identification of Helicobacter spp. and to determine the occurrence of these species in dogs. Twenty-three out of 25 dogs (92%) were positive for Helicobacter-like organisms in cytological screening. Culture was successful from biopsies of 5/25 dogs. The isolates were analyzed by electron microscopy, biochemical and physiological tests, whole protein analysis and 16S rDNA sequencing. Helicobacter felis was identified in four samples and Helicobacter bizzozeronii in one sample. Only the whole protein analysis in combination with electron microscopy was able to clearly discriminate the two species. Compared to the high prevalence of Helicobacter-like organisms, the occurrence of H. felis and H. bizzozeronii, was low (17 and 4%, respectively). No Flexispira rappini-like organisms or H. salomonis were detected. Electron microscopy revealed that H. bizzozeronii-like microorganisms were present in three additional biopsies where we were unable to culture any Helicobacter-like organisms. These observations indicate that in the stomach of dogs not all helicobacters are culturable. The unculturable bacteria appeared to be the prevalent ones and may represent different spiral organisms. The presence of distinct helicobacters with different characteristics can reflect different roles in the pathogenesis of canine gastric disease.     

犬源大肠杆菌耐药与毒力的分子特征

从9份病犬血便和6份健康犬粪便样品中分离出17株大肠杆菌,分别进行了生化鉴定,耐药基因blaCTX-M、blaTEM和blaSHV以及24种毒力基因的PCR扩增,13种抗菌药的药敏检测,以及ESBL表型鉴定、MLST分型、菌株分群和质粒分型。研究发现,病犬分离出的大肠杆菌多为致病性的ESBL表型耐药菌,携带多种质粒以及blaCTX-M和/或blaTEM-1基因,检测到10种毒力相关基因,健康犬分离出的大肠杆菌对多数抗菌药敏感,绝大部分未检测到质粒及blaCTX-M、blaTEM和blaSHV耐药基因。本研究从健康犬大肠杆菌分离株检测到3种新的ST型,分别为ST4001、ST4002和ST4003。结果表明,犬致病性大肠杆菌耐药情况十分严重,其临床治疗难度以及耐药性传播风险将加大,并对于人类健康具有潜在威胁,应予以重视。     

Phenotypic characterization and in vitro antifungal sensitivity of Candida spp. and Malassezia pachydermatis strains from dogs

Yeasts of the genera Candida and Malassezia can be found as commensal microorganisms in animals. The main species of importance in veterinary medicine are Malassezia pachydermatis and Candida albicans. The objectives of this study were to conduct a phenotypic characterization and to evaluate the in vitro antifungal sensitivity of strains of C. albicans (n=5), C. tropicalis (n=3) and M. pachydermatis (n=32) isolated from dogs. The phenotyping was based on macro and micromorphological features as well as biochemical analysis. The techniques of microdilution in broth and dilution in agar were used to evaluate the in vitro sensitivity of Candida spp. and M. pachydermatis, respectively. The tested drugs were ketoconazole (KTC), itraconazole (ITC), fluconazole (FLC) and amphotericin B (AMB). The morphological analysis of the strains of Candida spp. and M. pachydermatis did not show any noteworthy alterations when compared to standard strains. On the other hand, in the biochemical tests, 34.4% of the strains of M. pachydermatis were negative for the urease test. Four strains of C. albicans were resistant to FLC with a minimum inhibitory concentration (MIC) >64microg/mL and all were resistant to KTC and ITC (MIC>16microg/mL). The MIC for two strains of C. tropicalis were >16microg/mL for KTC and ITC, and >64microg/mL for FLC. It is worth highlighting that all of the strains tested were sensitive to AMB with the MIC varying from 0.25-1.0microg/mL. All strains of M. pachydermatis were sensitive to ITC with a minimum fungistatic concentration (MFC) 0.0075microg/mL. The MIC for 29 strains was the same (MFC0.0075microg/mL) for KTC. The MFCs for FLC varied from 1 to 16microg/mL, and for AMB, the MFC interval was 0.125-8microg/mL. There were no alterations in the classic phenotypic features of the strains of Candida spp. and M. pachydermatis isolated from dogs but, unlike M. pachydermatis, Candida spp. were much more resistant to azole antifungal agents.     

Molecular characterization of Cryptosporidium in Brazilian sheep

Feces were collected from 125 sheep between January and December 2007, on ten farms in the State of Rio de Janeiro, Brazil, and examined for the presence of Cryptosporidium. Ninety samples were collected from lambs 2 to 6 months of age, and 35 were from sheep over 12 months of age. All samples were subjected to molecular analysis by polymerase chain reaction (nested PCR) in two steps of the SSU rRNA. Two samples (1.6%) from the lambs were positive, and after sequencing were identified as Cryptosporidium ubiquitum. This species has been reported worldwide and it is considered a zoonotic pathogen since it has been found and in several animal species and humans. However, because of the low frequency of C. ubiquitum found, the risk for public health in this region may not be high.     

Molecular detection and characterization of Ehrlichia canis from dogs in Turkey

Seroprevalence of Ehrlichia canis antibodies among dogs in Turkey were previously reported, however, the ehrlichial organism has never been characterized in this region. The current study examined dogs from Ankara with febrile illness for E. canis infection with E. canis-specific PCR. Three of the 12 blood specimens from dogs showing clinical signs compatible with canine ehrlichiosis were found to be positive by PCR using E. canis-specific primers. E. canis detected in one of the blood specimens was designated as Kutahya strain. The representative E. canis strain was characterized by 16S rRNA gene sequencing and Western blot analysis of the plasma sample from the dog infected with E. canis. The 16S rRNA sequence (1,388 bp) of the E. canis Kutahya was identical to that of Ehrlichia ovina from a sheep in Turkey and Venezuelan Dog Ehrlichia (VDE) and was closely related (99.9%) to that of type strain of E. canis, Oklahoma. The plasma of the dog infected with E. canis Kutahya was analyzed by Western blotting using the purified E. canis Oklahoma strain as antigen. The reactive antibody profiles of the dog infected with E. canis Kutahya was found to be similar to those of dogs infected with E. canis Oklahoma and VDE, suggesting the antigenic similarities among these strains. The findings in this study would help for a better understanding of epidemiology of canine ehrlichiosis. This is the first report of molecular detection and characterization of an ehrlichial agent in Turkey.