Anna-Mari Bosman

Babesia lengau sp. nov., a Novel Babesia Species in Cheetah (Acinonyx jubatus, Schreber, 1775) Populations in South Africa

ABSTRACT In a previous paper, we reported on a large number of cheetah blood specimens that gave positive signals only for Babesia and/or Theileria genus-specific probes on the reverse line blot (RLB) assay, indicating the presence of a novel species or variant of an existing species. Some of these specimens were investigated further by microscopic, serological, sequencing, and phylogenetic analyses. The near-full-length 18S rRNA genes of 13 samples, as well as the second internal transcribed spacer (ITS2) region, were amplified, cloned, and sequenced. A species-specific RLB probe, designed to target the hypervariable V4 region of the 18S rRNA gene for detection of the novel Babesia sp., was used to screen an additional 137 cheetah blood specimens for the presence of the species. The prevalence of infection was 28.5%. Here we describe the morphology and phylogenetic relationships of the novel species, which we have named Babesia lengau sp. nov.

Babesia lengau associated with cerebral and haemolytic babesiosis in two domestic cats

BackgroundAlthough reported sporadically from various countries, feline babesiosis appears to be a significant clinical entity only in South Africa, where Babesia felis is usually incriminated as the causative agent. Babesia lengau, recently described from asymptomatic cheetahs, has now possibly been incriminated as the causative agent in two severe clinical cases in domestic cats.FindingsBoth cats were euthanised in extremis. While typical feline babesiosis in South Africa is an afebrile disease with a chronic manifestation, there was acute onset of severe clinical signs in both cats and their body temperatures were above the normal range when they were presented for treatment. Haemolytic anaemia was confirmed in one case. To our knowledge, this is the first report of cerebral babesiosis in cats.On reverse line blot 18S rDNA PCR products obtained from both cats showed positive hybridization profiles with the B. lengau species-specific probe. The two partial parasite 18S rRNA gene sequences obtained, showed high sequence similarity (99.9%) to B. lengau. In a representative tree constructed by the neighbor-joining method using the two-parameter model of Kimura the two obtained partial 18S rDNA sequences and that of B. lengau formed a monophyletic group with B. conradae and sequences previously isolated from humans and wildlife in the western USA.ConclusionAll clinical cases of feline babesiosis in South Africa are not necessarily caused by B. felis. Other piroplasms, e.g. B. lengau, may be incriminated in clinical cases, especially those occurring outside the known endemic area.

Tick-borne blood parasites in nyala (Tragelaphus angasii, Gray 1849) from KwaZulu-Natal, South Africa

A total of 97 blood samples of nyala (Tragelaphus angasii, Gray 1849) from South Africa were tested for the presence of tick-borne haemoparasites by means of polymerase chain reaction (PCR) and reverse line blot (RLB) hybridization. The majority of blood samples contained several different haemoparasites, often in combination. Prevalent haemoparasites were Theileria sp. (kudu), T. buffeli, Theileria sp. (sable), T. bicornis, Ehrlichia sp. Omatjenne, Anaplasma marginale and A. bovis. This serves as the first report of Theileria sp. (kudu), T. buffeli, T. bicornis, Ehrlichia sp. Omatjenne, A. marginale and A. bovis in nyala, who seem to carry multiple haemoparasites without ill effect.

Genetic analysis of the VP2-encoding gene of canine parvovirus strains from Africa

Since the emergence of canine parvovirus type-2 (CPV-2) in the early 1970s, it has been evolving into novel genetic and antigenic variants (CPV-2a, 2b and 2c) that are unevenly distributed throughout the world. Genetic characterization of CPV-2 has not been documented in Africa since 1998 apart from the study carried out in Tunisia 2009. A total of 139 field samples were collected from South Africa and Nigeria, detected using PCR and the full length VP2-encoding gene of 27 positive samples were sequenced and genetically analyzed. Nigerian samples (n=6), South Africa (n=19) and vaccine strains (n=2) were compared with existing sequences obtained from GenBank. The results showed the presence of both CPV-2a and 2b in South Africa and only CPV-2a in Nigeria. No CPV-2c strain was detected during this study. Phylogenetic analysis showed a clustering not strictly associated with the geographical origin of the analyzed strains, although most of the South African strains tended to cluster together and the viral strains analyzed in this study were not completely distinct from CPV-2 strains from other parts of the world. Amino acid analysis showed predicted amino acid changes.

Transmembrane proteins - mining the cattle tick transcriptome

Managing the spread and load of pathogen-transmitting ticks is an important task worldwide. The cattle tick, Rhipicephalus microplus, not only impacts the economy through losses in dairy and meat production, but also raises concerns for human health in regards to the potential of certain transmitted pathogens becoming zoonotic. However, novel strategies to control R. microplus are hindered by lack of understanding tick biology and the discovery of suitable vaccine or acaricide targets. The importance of transmembrane proteins as vaccine targets are well known, as is the case in tick vaccines with Bm86 as antigen. In this study, we describe the localization and functional annotation of 878 putative transmembrane proteins. Thirty proteins could be confirmed in the R. microplus gut using LC-MS/MS analysis and their roles in tick biology are discussed. To the best of our knowledge, 19 targets have not been reported before in any proteomics study in various tick species and the possibility of using the identified proteins as targets for tick control are discussed. Although tissue expression of identified putative proteins through expansive proteomics is necessary, this study demonstrates the possibility of using bioinformatics for the identification of targets for further evaluation in tick control strategies.

Molecular characterisation of Newcastle disease virus isolates from different geographical regions in Mozambique in 2005

Newcastle disease (ND) is regarded as a highly contagious and economically important disease in poultry and has a worldwide distribution. Viral determinants for Newcastle disease virus (NDV) virulence are not completely understood and viruses of different pathotypes can be found at live-bird markets in different geographical areas. The prevalence of Newcastle disease in village poultry in Mozambique is not well documented and strains of NDV involved in yearly outbreaks are unknown. The fusion (F) protein is an important determinant of pathogenicity of the virus and is used commonly for phylogenetic analysis. Newcastle disease viruses from various geographical regions of Mozambique were sequenced and compared genetically to published sequences obtained from GenBank. Samples were collected in three different areas of Mozambique and NDV was isolated by infection of embryonated chicken eggs. Sequence analysis of the F-protein encoding gene was used to classify 28 isolates from Mozambique into genotypes and compare these genotypes phylogenetically with existing genotypes found in GenBank. The isolates obtained from Mozambique grouped mainly into two clades. In the first clade, 12 isolates grouped together with sequences of isolates representing genotypes from Mozambique that were previously described. In the second clade, 16 isolates group together with sequences obtained from GenBank originating from Australia, China, South Africa and the USA. Eleven of these isolates showed a high similarity with sequences from South Africa. The number of samples sequenced (n = 28), as well as the relatively small geographical collection area used in this study, are too small to be a representation of the circulating viruses in Mozambique in 2005. Viruses characterised in this study belonged to lineage 5b, a similar finding of a previous study 10 years ago. From this data, it merely can be concluded that no new introduction of the virus occurred from 1995 to 2005 in Mozambique.

SENSITIVITY AND SPECIFICITY OF A NESTED POLYMERASE CHAIN REACTION FOR DETECTION OF LENTIVIRUS INFECTION IN LIONS (PANTHERA LEO)

Abstract Feline immunodeficiency virus (FIV) is a lentivirus in the Retroviridae family that causes lifelong infection in domestic cats. The lentivirus of African lions (Panthera leo), referred to as FIVple, is endemic in certain lion populations in eastern and southern Africa. Lentivirus infection leads to immunologic dysfunction and immunosuppressive disease in domestic cats; however, little is known about the pathogenic effects of infection in lions, nor about the epidemiologic impact on free-ranging and captive populations. Whole blood and serum samples were collected opportunistically from free-ranging lions in Kruger National Park, Republic of South Africa (RSA). Whole blood and serum samples were also collected from captive wild lions in the RSA. A nested polymerase chain reaction (PCR) assay for detection of FIV was performed on all whole blood samples. In addition, serum samples were tested for cross-reactive antibodies to domestic feline lentivirus antigens and puma lentivirus synthetic envelope peptide antigen. The PCR assay successfully amplified the lion lentivirus from African lions. The relative sensitivity and relative specificity were 79% and 100%, respectively, and the positive and negative predictive values were 100% and 67%, respectively. This research represents the first study to compare genetic material with antibody-based methods of lentivirus detection on lions in RSA. Using PCR as an additional diagnostic test for FIV in lions will increase screening sensitivity and will allow viral characterization among circulating isolates and monitoring of changes in the viral epidemiology within geographic regions and populations over time.

Molecular characterisation of Mycoplasma species isolated from the genital tract of Dorper sheep in South Africa

Biochemical and molecular analysis were conducted on 34 strains of Mycoplasma species isolated between 2003 and 2009 from the genital tract of clinically healthy Dorper sheep and sheep with ulcerative vulvitis and balanitis. Earlier publications identified the causative agent as Mycoplasma mycoides mycoides large colony (MmmLC) and Arcanobacterium pyogenes. The aims of the study were to characterise Mycoplasma species isolated from the genital tract of Dorper sheep with polymerase chain reaction assay, cloning and gene sequencing. Basic Local Alignment Search Tool (BLAST) results revealed six predominant Mycoplasma species: Mycoplasma arginini, Mycoplasma bovigenitalium, Arcanobacterium laidlawii, MmmLC, Mycoplasma sp. ovine/caprine serogroup II and M. canadense. Sequencing of the 34 isolates were analysed using phylogenetic methods, and 18 (50%) were identified as M. arginini with 99% - 100% similarity to M. arginini from England and Sweden. Six isolates showed 99% similarity to M. bovigenitalium strains from Turkey and Germany. Two isolates had 99% similarity to an M. sp. ovine/caprine sero group II from the United Kingdom. BLAST for two isolates revealed 99% similarity to Acholeplasma laidlawii from India, another two were 99% similar to MmmLC strain from Sweden, two showed 98% similarity to Mycoplasma sp. Usp 120 from Brazil, and two isolates have a 97% - 99% similarity to M. mm. Jcv1 strain from the United States of America. Finally, one isolate showed similarity of 99% to Mycoplasma canadense strain from Italy. The findings support the hypothesis that ulcerative vulvitis and balanitis of Dorper sheep in South Africa (SA) is a multifactorial disease with involvement of different Mycoplasma species.Biochemical and molecular analysis were conducted on 34 strains of Mycoplasma species isolated between 2003 and 2009 from the genital tract of clinically healthy Dorper sheep and sheep with ulcerative vulvitis and balanitis. Earlier publications identified the causative agent as Mycoplasma mycoides mycoides large colony (MmmLC) and Arcanobacterium pyogenes. The aims of the study were to characterise Mycoplasma species isolated from the genital tract of Dorper sheep with polymerase chain reaction assay, cloning and gene sequencing. Basic Local Alignment Search Tool (BLAST) results revealed six predominant Mycoplasma species: Mycoplasma arginini, Mycoplasma bovigenitalium, Arcanobacterium laidlawii, MmmLC, Mycoplasma sp. ovine/caprine serogroup II and M. canadense. Sequencing of the 34 isolates were analysed using phylogenetic methods, and 18 (50%) were identified as M. arginini with 99% – 100% similarity to M. arginini from England and Sweden. Six isolates showed 99% similarity to M. bovigenitalium strains from Turkey and Germany. Two isolates had 99% similarity to an M. sp. ovine/caprine sero group II from the United Kingdom. BLAST for two isolates revealed 99% similarity to Acholeplasma laidlawii from India, another two were 99% similar to MmmLC strain from Sweden, two showed 98% similarity to Mycoplasma sp. Usp 120 from Brazil, and two isolates have a 97% – 99% similarity to M. mm. Jcv1 strain from the United States of America. Finally, one isolate showed similarity of 99% to Mycoplasma canadense strain from Italy. The findings support the hypothesis that ulcerative vulvitis and balanitis of Dorper sheep in South Africa (SA) is a multifactorial disease with involvement of different Mycoplasma species.

Black Rhinoceros (Diceros bicornis) Populations in Northwestern Namibia Are Apparently Not Infected with Piroplasms

Babesiosis is a potentially fatal disease in black rhinoceroses. Blood specimens collected from black rhinoceroses from Etosha National Park (n=29) and Damaraland (n=22), Namibia, were subjected to polymerase chain reaction using Theileria and Babesia genus-specific primers and reverse line blot, with negative results. The animals were sparsely infested with ticks. In the absence of suitable prophylactic measures, naı¨ve rhinoceroses would be at risk if translocated to Babesia-endemic areas.

BACTERIAL PROFILE OF NECROTIC PULPS IN CHEETAH (ACINONYX JUBATUS) CANINE TEETH

Abstract The role of microbes and their antimicrobial susceptibilities in both acute and chronic infections of the dental pulp in humans has been well studied. Presently, no data are available on endodontic pathogens in cheetahs (Acinonyx jubatus). The aim of this study was to isolate and identify the bacteria found in the canine teeth of cheetahs, where the pulp was necrotic and exposed due to a complicated crown fracture. Thirty-six microbiologic samples were taken from root canals (RCs) of the canine teeth of 19 cheetahs: one pulp sample was taken from 10 cheetahs, four samples from 2 cheetahs, two samples from 3 cheetahs, and three samples from 4 cheetahs. Exposed pulps were cultured for aerobic and anaerobic bacteria; an additional screening with a 16S rRNA–specific polymerase chain reaction (PCR) was used for the last six samples. Antimicrobial susceptibility of isolates was determined by use of the Kirby–Bauer diffusion test. In total, 59 cultivable isolates belonging to 19 microbial species and 13 genera were recovered from the 36 RCs sampled. Only two samples yielded no cultivable bacteria. Thirty-two (54.49%) of the cultivable isolates were Gram positive and 27 (45.71%) were Gram negative. The maximum number of isolates cultivated from an individual RC was six. Facultative anaerobes (62.72%) were the most common bacteria of the RCs that yielded cultivable bacteria. Of the isolates, 28.81% were aerobic and 8.47% were strict anaerobes. The antimicrobials that showed the greatest efficacy in vitro against the different bacteria isolates were amikacin and gentamicin. The more common bacterial species isolated by PCR were anaerobes (60.8%), facultative anaerobes (30.2%), and aerobes (8.6%).