Emergence of Theileria species in ticks from free-ranging domestic animals in Raymond Mhlaba local municipality, South Africa.

Ticks infestation and diseases associated with it, are becoming a major life threatening concern to wildlife, domesticated animals and human health in general. Besides causing skin damage, ticks infestations have become a growing burden in food security and transmission of multiple pathogens. There is paucity of data on the occurrence of etiologic agents of tick-borne diseases in the Eastern Cape Province South Africa. We therefore carried out a molecular surveillance on Babesia and Theileria species in ticks obtained from livestock in Raymond Mhlaba District Municipality of the Province. A total of 962 ticks were collected and were morphologically identified and processed for DNA extraction using commercial DNA extraction kit. The extracted DNA samples were used to molecular identification of the ticks, and also to assess the occurrence of the Babesia and Theileria spp by PCR using genus specific primers. Positive amplicons obtained were sequenced, processed and characterised using appropriate bioinformatics tools. The molecular and morphological identifications of ticks obtained from the domestic animals in the study areas revealed that they belong to three different genera namely: Haemophalis, Rhipicephalus, and Amblyomma in ascending order of their abundance. Furthermore, the DNA of Theileria spp. was detected from 10 out of 962 ticks screened, with an overall infection of about 1% for Rhipicephalus spp., while none of the ticks was positive for Babesia spp. The phylogenetic analysis of the 10 theilerial sequences showed that nine (9) clustered distinctly within the T. orientalis complex clade, while only one (1) sequence formed a cluster with reference sequences of T. velifera. The findings from this study therefore expand the knowledge on recent emergence of Theileria spp. in livestock reared in the study area. This calls for an urgent effort in curbing the further spread of the pathogens in the area and beyond.

Genomic Profiling for Piroplasms in Feeding Ixodid Ticks in the Eastern Cape, South Africa.

Importation of tick-infected animals and the uncontrollable migration of birds and wild animals across borders can lead to geographical expansion and redistribution of ticks and pathogen vectors, thus leading to the emergence and re-emergence of tick-borne diseases in humans and animals. Comparatively, little is known about the occurrence of piroplasms in ixodid ticks in the Eastern Cape, South Africa, thus necessitating this study, which is aimed at detecting piroplasms (Theileria and Babesia) from feeding tick samples collected from cattle, sheep, and goats in selected sites in the Eastern Cape, South Africa. A total of 1200 feeding ixodid ticks collected from farm animals at selected homesteads were first subjected to molecular identification using mitochondrial 12S ribosomal RNA (rRNA) gene by PCR and were further tested for the presence of piroplasms through amplification of the 18S rRNA gene via nested-PCR followed by sequencing of the PCR products. The results indicated that 853 (71.1%) corresponded to the genus Rhipicephalus, 335 (27.9%) corresponded to genus Amblyomma, and 12 (1%) corresponded to genus Haemaphysalis. Amblyomma hebraeum and Rhipicephalus appendiculatus were the most common identified ticks from this study. The 18S rRNA nested-PCR revealed that 44 (3.7%) samples were confirmed positive for Theileria. A homology search for the generated sequences revealed a high percentage identity of 98-98.9% similarity to T. buffeli, T. orientalis, and T. sergenti in the GenBank. Based on the results obtained herein, we conclude that there is a big diversity of Theileria species; therefore, we suggest that this research should cover more geographical areas in order to reveal the true prevalence of this pathogen in the studied area because this will be a great step in the possible prevention of an outbreak that could have devastating effects on livestock production and human health in both the studied areas and South Africa at large.

Chemokine Coreceptor Usage Among HIV-1 Drug-Naive Patients Residing in the Rural Eastern Cape, South Africa.

Sub-Saharan region in Africa still holds the highest burden of HIV/AIDS globally. HIV-1 requires coreceptor to gain entry into permissive cells to initiate infection. Molecular analysis of the chemokine coreceptor usage is important clinically and in the effective management of AIDS virus. This study aims to determine the coreceptor usage among HIV-1 drug-naive patients residing in the rural Eastern cape, South Africa. We collected blood samples from 55 HIV-infected patients into an anticoagulant vacutainer. RNA was extracted from separated plasma, and reverse transcription-polymerase chain reaction (RT-PCR) was performed followed by nested polymerase chain reaction to amplify the partial envelope fragment spanning the C2-C3 region. Sanger sequencing was done on the amplicons using the BigDye Terminator V3.1 sequencing kit (Applied Biosystems, Foster City, CA) while sequences were manually edited using BioEdit and Geneious 10.2.6 tools. The WebPSSM and Geno2pheno online tools were also utilized to predict coreceptor tropism while the phylogenetic analysis of the isolates was determined using MEGA 7. Of the 55 blood samples collected for the study, 50 (91%) were successfully amplified and sequenced. The mean age of the patients was 32 (18-56) years while the ratio of men to women was 35% and 65% correspondingly. Phylogenetic analysis revealed that all 50 sequences clustered with HIV-1 subtype C reference strains. Viral tropism of the V3 loop revealed 47 sequences to be R5 strains, while three sequences (T1E, T10E, and T11E,) were classified as X4 strains based on the WebPSSM and the Geno2pheno algorithm. HIV-1 R5 tropic strains were the most dominant virus obtained from this study, while HIV-1 subtype C still drives the epidemic in South Africa suggesting greater in vivo and host pathogen fitness. Documented data on mapping out cellular tropism based on viral tropism are important as maraviroc and the other CCR5 antagonist could be introduced as part of the treatment regimen in South Africa.

Phylogenetic profiling for zoonotic Ehrlichia spp. from ixodid ticks in the Eastern Cape, South Africa.

Ticks are obligate hematophagous parasite of vertebrate that transmit a range of pathogenic microorganisms that can cause diseases in livestock and humans. The range of tick-borne disease causative agents infecting domestic animals and humans has recently increased. Several significant zoonotic tick-borne diseases such as ehrlichiosis among others are on the increase worldwide. This study was designed to investigate the occurrence of zoonotic Ehrlichia spp. from samples collected from livestock in selected communities in the Eastern Cape Province, South Africa. Tick samples were manually collected from domesticated animals in selected homesteads. The ticks were morphologically identified to species and tested for Ehrlichia infection via polymerase chain reaction (PCR), using genus-specific disulphide bond formation protein (dsbA) gene primers. This was followed by sequence analysis of amplicons and phylogeny. Of the 1,200 ticks collected, Amblyomma hebraeum was most prevalent (n = 335; 27.9%), followed by Rhipicephalus appendiculatus (n = 274; 22.8%), Rhipicephalus decoloratus; (n = 224; 18.7%) and Rhipicephalus eversti eversti (n = 200, 16.7%). Ehrlichia DNA was detected in 19/1,200 (1.6%) of the screened DNA samples. A homology search of the generated sequences revealed a high percentage of identity between 95% and 98% with other homologous dsbA gene sequences of other Ehrlichia species in GenBank. Phylogenetic analysis showed that the obtained sequences clustered unambiguously with other Ehrlichia sequences from different geographical regions of the world. We concluded that Ehrlichial pathogens are vectored by the ticks collected from domesticated animals in the study areas, thus suggesting concern for public health, as some of the recovered pathogens are zoonotic in nature and could pose serious public health risk through human exposure to tick bites.

Molecular Genetics and the Incidence of Transmitted Drug Resistance Among Pre-Treatment HIV-1 Infected Patients in the Eastern Cape, South Africa.

BACKGROUND: Transmitted drug resistance (TDR) remains a significant threat to Human immunodeficiency virus (HIV) infected patients that are not exposed to antiretroviral treatment. Although, combined antiretroviral therapy (cART) has reduced deaths among infected individuals, emergence of drug resistance is gradually on rise. OBJECTIVE: To determine the drug resistance mutations and subtypes of HIV-1 among pre-treatment patients in the Eastern Cape of South Africa. METHODS: Viral RNA was extracted from blood samples of 70 pre-treatment HIV-1 patients while partial pol gene fragment amplification was achieved with specific primers by RT-PCR followed by nested PCR and positive amplicons were sequenced utilizing ABI Prism 316 genetic sequencer. Drug resistance mutations (DRMs) analysis was performed by submitting the generated sequences to Stanford HIV drug resistance database. RESULTS: Viral DNA was successful for 66 (94.3%) samples of which 52 edited sequences were obtained from the protease and 44 reverse transcriptase sequences were also fully edited. Four major protease inhibitor (PI) related mutations (I54V, V82A/L, L76V and L90M) were observed in seven patients while several other minor and accessory PIs were also identified. A total of 11(25.0%) patients had NRTIs related mutations while NNRTIs were observed among 14(31.8%) patients. K103N/S, V106M and M184V were the most common mutations identified among the viral sequences. Phylogenetic analysis of the partial pol gene indicated all sequences clustered with subtype C. CONCLUSION: This study indicates that HIV-1 subtype C still predominates and responsible for driving the epidemic in the Eastern Cape of South Africa with slow rise in the occurrence of transmitted drug resistance.

Increasing diversity of swine parvoviruses and their epidemiology in African pigs.

Detection of infectious viral agents has been on the increase globally with the advent and usage of more sensitive and selective novel molecular techniques in the epidemiological study of viral diseases of economic importance to the swine industry. The observation is not different for the pig-infecting member of the subfamily Parvovirinae in the family Parvoviridae as the application of novel molecular methods like metagenomics has brought about the detection of many other novel members of the group. Surprisingly, the list keeps increasing day by day with some of them possessing zoonotic potentials. In the last one decade, not less than ten novel swine-infecting viruses have been added to the subfamily, and ceaseless efforts have been in top gear to determine the occurrence and prevalence of the old and new swine parvoviruses in herds of pig-producing countries worldwide. The story, however, is on the contrary on the African continent as there is presently a dearth of information on surveillance initiatives of the viruses among swine herds of pig-producing countries in the region. Timely detection and characterization of the viral pathogens is highly imperative for the implementation of effective control and prevention of its spread. This review therefore presents a concise overview on the epidemiology of novel porcine parvoviruses globally and also provides up-to-date highlights on the reported cases of the viral agents in the African sub-region.

Prevalence of porcine parvoviruses in some South African swine herds with background of porcine circovirus type 2 infection.

The classical porcine parvovirus is an important pathogen of reproductive disorders in pigs with a confirmed history of global distribution. The detection of many novel porcine parvoviruses has however been on the increase for the past few years, but there is a dearth of information on the occurrence and prevalence of these viruses in South Africa. Molecular detection of some known parvoviruses, namely porcine parvoviruses (PPVs) - 1, 2, 3 and 4, porcine bocavirus-like virus (PBo-likeV) and porcine bocaviruses (PBoV1/2), was carried out from 110 randomly selected archived swine samples collected in the year 2015 and 2016. Samples were drawn from previously screened and confirmed porcine circovirus type 2 (PCV2) infected farms, with farm-level occurrence ranged from 5.6 to 60%. The findings showed that all the screened parvoviruses were present as follows: PPV1 (29.1%), PPV2 (21.8%), PPV3 (5.5%), PPV4 (43.6%), PBo-likeV (21.8%) and PBoV1/2 (44.6%). The frequency of double infections of the viruses was as high as 18.2% of PPV2/PPV4 and PPV4/PBoVs; while 17.3% and 7.3% of the screened samples showed multiple infections of the three and four viruses respectively. Further phylogenetic analyses of partial PPV1, 2 and PBoV1/2 sequences showed two major clades for each of the viruses. This study reports the first epidemiological survey and molecular characterisation of the classical and emerging porcine parvoviruses in South African swine herds. It also gives insights into the diversity and distribution of these viral pathogens within the herds of the study area and confirms their co-infection potentials with PCV2.

Molecular detection of Porcine circovirus type 2 in swine herds of Eastern Cape Province South Africa.

BACKGROUND: Porcine circovirus type 2 (PCV2) remains the main causative viral pathogen of porcine circovirus-associated diseases (PCVAD) of great economic importance in pig industry globally. This present study aims at determining the occurrence of the viral pathogen in swine herds of the Province. RESULTS: The data obtained revealed that 15.93% of the screened samples (54/339) from the swine herds of the studied areas were positive for PCV2; while the severity of occurrence of the viral pathogen as observed at farm level ranges from approximately 5.6 to 60% in the studied farms. The majority (15 out of 17 = 88%) of the analyzed sequences were found clustering with other PCV2b strains in the phylogenetic analysis. More interestingly, two other sequences obtained were also found clustering within PCV2d genogroup, which is presently another fast-spreading genotype with observable higher virulence in global swine herds. CONCLUSION: This is the first report of PCV2 in swine herds of the Province and the first detection of PCV2b and PCV2d in South African swine herds. It follows the first reported case of PCV2a in an outbreak of porcine multisystemic wasting syndrome (PMWS) in Gauteng Province, South Africa more than one decade ago. This finding confirmed the presence of this all-important viral pathogen in pigs of the region; which could result in a serious outbreak of PCVAD and huge economic loss at the instances of triggering factors if no appropriate measures are taken to effectively curb its spread.

Chemical constituents, antibacterial and antioxidant properties of the essential oil flower of Tagetes minuta grown in Cala community Eastern Cape, South Africa.

BACKGROUND: Tagetes minuta has a long record of human use for the treatment of stomach and intestinal diseases. Most drugs used for diseases treatment are less efficacious with side effects and this brought the search for new treatment regimens mainly from medicinal plants. METHOD: The essential oil (EO) was extracted by Clevenger's-type apparatus and its chemical composition, antioxidant and antibacterial properties were determined by GC-MS, spectrophotometric and broth dilution methods respectively. S. uberis, E. cloacae, S. aureus, M. smegmatis, L. ivanovii, Vibrio spp. and E. coli bacteria strains were used as test bacteria. RESULTS: GC-MS analysis revealed 98 compounds in the EO flower of T. minuta and ß-Ocimene (14. 40%) was the major chemical constituents. The EO exhibited highest inhibitory effect against DPPH radical, followed by its effect on ABTS, while LP radical showed the least sensitivity with IC50 values of 2.45 mg/mL, 2.76 mg/mL and 3.23 mg/mL respectively. The EO showed antibacterial activities against all test organisms with MIC value for S. aureus, M. smegatis and S. uberis at 0.125 mg/mL and for L. ivanovii, Vibrio spp., E. cloacae and E. coli at 0.06 mg/mL. The EO showed MBC against E. cloacae and E. coli at 0.06 mg/mL at 0.5 mg/mL for S. uberis and 0.125 mg/mL for Vibrio spp. CONCLUSION: Findings from this study suggest that the EO of T. minuta flower may be a useful candidate in the search for lead constituents for the synthesis of new potent antibacterial and antioxidant agent.

Incidence and Molecular Characterization of Hepatitis E Virus from Swine in Eastern Cape, South Africa.

Hepatitis E virus-mediated infection is a serious public health concern in economically developing nations of the world. Globally, four major genotypes of HEV have been documented. Hepatitis E has been suggested to be zoonotic owing to the increase of evidence through various studies. Thus far, this paper reports on prevalence of hepatitis E virus among swine herd in selected communal and commercial farms in the Eastern Cape Province of South Africa. A total of 160 faecal samples were collected from swine herds in Amathole and Chris Hani District Municipalities of Eastern Cape Province for the presence of HEV. Of the 160 faecal samples screened, only seven were positive (4.4%) for HEV. The nucleotide sequences analyses revealed the isolates as sharing 82% to 99% identities with other strains (KX896664, KX896665, KX896666, KX896667, KX896668, KX896669, and KX896670) from different regions of the world. We conclude that HEV is present among swine in the Eastern Cape Province, albeit in low incidence, and this does have public health implications. There is a need for maintenance of high hygienic standards in order to prevent human infections through swine faecal materials and appropriate cooking of pork is highly advised.

Multidrug-Resistant Salmonella Isolates from Swine in the Eastern Cape Province, South Africa.

The exposure of farm animals to antimicrobials for treatment, prophylaxis, or growth promotion can select for resistant bacteria that can be transmitted to humans, and Salmonella as an important zoonotic pathogen can act as a potential reservoir of antimicrobial resistance determinants. We assessed the antibiogram profiles of Salmonella species isolated from pig herds in two commercial farms in South Africa. Two hundred fifty-eight presumptive Salmonella isolates were recovered from the fecal samples of 500 adult pigs. Specific primers targeting Salmonella serogroups A, B, C1, C2, and D were used to determine the prevalence of different serogroups. Only serogroup A (n = 48) was detected, while others were not. Antimicrobial susceptibility of the confirmed Salmonella serogroup A isolates was performed by using the disk diffusion method against a panel of 18 antibiotics. All the 48 isolates were resistant to tetracycline and oxytetracycline, while 75% were resistant to ampicillin, sulphamethoxazole-trimethoprim, nalidixic acid, and streptomycin. All the isolates exhibited multidrug resistance, with the predominant phenotype being against 11 antibiotics, and multiple antibiotic resistance index ranged between 0.3 and 0.6. The incidence of genes encoding resistance against ampicillin (ampC), tetracycline (tetA), and streptomycin (strA) were 54, 61, and 44%, respectively. We conclude that healthy pigs are potential reservoirs of multidrug-resistant Salmonella that could be transmitted to humans through the food chain and, hence, a significant public health threat.

Occurrence of non-O157 Shiga toxin-producing Escherichia coli in two commercial swine farms in the Eastern Cape Province, South Africa.

Shiga toxin-producing Escherichia coli (STEC) is one of the most significant causes of food-borne infections capable of causing serious health complications in humans. Even though ruminants are known to be the major reservoirs of STEC, other non-ruminant food producing animals may also harbour pathogenic E. coli strains. In this study, we investigated the prevalence of E. coli serogroups O26, O111, O121, O145, and O157 and their associated virulence genes (stx1, stx2, eae, and ehxA) in swine faecal samples obtained from the two major commercial farms located in the Nkonkobe Municipality, Eastern Cape, South Africa. The proportions of serogroups detected were O26; 35 (7%), O145; 14 (2.8%), and O157:H7; 43 (8.6%) of the total animals sampled. Out of the 500 animals sampled, 22 isolates of E. coli (1.4%) tested positive for the stx2 gene, and 7 of these isolates belonged to E. coli O26 serogroup, while the remaining 15 most likely belonged to serogroups untargeted in this study. Other virulence genes (stx1, eae, and ehxA) that we screened for were not detected. These findings reveal that pigs within the Eastern Cape Province of South Africa can harbour Shiga toxin-producing E. coli.

Antibiotic susceptibilities of enterococcus species isolated from hospital and domestic wastewater effluents in alice, eastern cape province of South Africa.

BACKGROUND: Antimicrobial resistance in microorganisms are on the increase worldwide and are responsible for substantial cases of therapeutic failures. Resistance of species of Enterococcus to antibiotics is linked to their ability to acquire and disseminate antimicrobial resistance determinants in nature, and wastewater treatment plants (WWTPs) are considered to be one of the main reservoirs of such antibiotic resistant bacteria. We therefore determined the antimicrobial resistance and virulence profiles of some common Enterococcus spp that are known to be associated with human infections that were recovered from hospital wastewater and final effluent of the receiving wastewater treatment plant in Alice, Eastern Cape. METHODS: Wastewater samples were simultaneously collected from two sites (Victoria hospital and final effluents of a municipal WWTP) in Alice at about one to two weeks interval during the months of July and August 2014. Samples were screened for the isolation of enterococci using standard microbiological methods. The isolates were profiled molecularly after targeted generic identification and speciation for the presence of virulence and antibiotic resistance genes. RESULTS: Out of 66 presumptive isolates, 62 were confirmed to belong to the Enterococcus genusof which 30 were identified to be E. faecalis and 15 E. durans. The remaining isolates were not identified by the primers used in the screening procedure. Out of the six virulence genes that were targeted only three of them; ace, efaA, and gelE were detected. There was a very high phenotypic multiple resistance among the isolates and these were confirmed by genetic analyses. CONCLUSIONS: Analyses of the results obtained indicated that hospital wastewater may be one of the sources of antibiotic resistant bacteria to the receiving WWTP. Also, findings revealed that the final effluent discharged into the environment was contaminated with multi-resistant enterococci species thus posing a health hazard to the receiving aquatic environment as these could eventually be transmitted to humans and animals that are exposed to it.