Molecular detection and genetic characterization of Mycoplasma gallisepticum and Mycoplasma synoviae in selected chicken breeds in South Africa.

BACKGROUND: The impact of chickens on maintaining the economy and livelihood of rural communities cannot be overemphasized. In recent years, mycoplasmosis has become one of the diseases that affect the success of South African chicken production. Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) are the most prevalent strains of Mycoplasma in South Africa. MG and MS are significant respiratory pathogens affecting the productivity of chickens. The present study aimed to molecularly detect using qPCR and characterize the presence of MG and MS using phylogenetic analysis. The phylogenetic analysis was utilized to clarify general evolutionary relationships between related taxa of different MG and MS observed in tracheal swabs from South African chicken breeds. METHODS: Forty-five tracheal swabs of the Lohmann Brown (n = 9), Rhode Island Red (n = 9), Ovambo (n = 9), Venda (n = 9), and Potchefstroom Koekoek (n = 9) breeds were collected from symptomatic chickens present in the commercial farm. To detect MG and MS, DNA was extracted from tracheal swabs and faecal samples, and qPCR was performed with a 16 s rRNA (310 bp) and vlhA (400 bp) gene fragment. Following the sequencing of all the amplicons, MG, and MS dendrograms showing the evolutionary relationships among the five South African chicken breeds and the GeneBank reference population were constructed. RESULTS: The qPCR revealed the presence of MG and MS in 22% (2/9) of the tracheal swab samples tested for MS only in Rhode Island Red breeds; 66.6% (6/9) and 33% (3/9) of the tested samples in Ovambo breeds; and 11.1% (1/9) and 44.4% (4/9) of the tested samples in Venda breeds. No MG or MS were detected in the Lohmann Brown or Potchefstroom Koekoek breed. Furthermore, qPCR revealed the presence of MG in pooled faecal samples from Lohmann Brown and Ovambo breeds. Eight different bacterial isolates were recognized from both samples. Four isolates were of the 16 s ribosomal ribonucleic acid (rRNA) gene (named PT/MG51/ck/00, PT/MG48/ck/00, PT/MG41/ck/00 and PT/MG71/ck/00) gene of Mycoplasma gallisepticum, and the other was Mycoplasma Synoviae variable lipoprotein hemagglutinin A (vlhA) gene (named PT/MSA22/ck/01, PT/MS41/ck/01, PT/MS74/ck/01 and PT/MS46/ck/01) which were available in GenBank. These isolates were successfully sequenced with 95-100% similarity to the isolates from the gene bank. CONCLUSION: The study revealed the presence of both MG and MS in the chicken breeds sampled. Furthermore, the different breeds of chicken were found to be susceptible to infection under the intensive or commercial management system. Therefore, continuous surveillance is encouraged to prevent the spread and outbreak of MG and MS in the poultry industry in South Africa.

A decontamination strategy for resolving SARS-CoV-2 amplicon contamination in a next-generation sequencing laboratory.

Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) amplicon contamination was discovered due to next-generation sequencing (NGS) reads mapping in the negative controls. Environmental screening was undertaken to determine the source of contamination, which was suspected to be evaporation during polymerase chain reaction (PCR) assays while using the coronavirus disease 2019 (COVID-19) ARTIC protocol. A decontamination strategy is hereby documented to assist laboratories that may experience similar amplicon contamination. Routine molecular laboratory environmental screening as a quality control is highly recommended.

Evidence of zoonotic rickettsiae in ixodid ticks of domestic animals in some communal farms in the Eastern Cape Province, South Africa.

Objective: The abundance of tick populations in South Africa represents a probable risk for both animal and human health. Rickettsia spp. and Borrelia spp. are well-known agents of emerging human tick-borne infectious diseases worldwide. Nevertheless, the epidemiology of their infections has been underreported in South Africa. Therefore, this study aimed to profile zoonotic Rickettsia and Borrelia species from ticks infesting domesticated animals in the Eastern Cape, South Africa. Materials and Methods: Morphological and molecular identification techniques were conducted on 1,200 tick samples collected from domestic animals before screening for the target bacterial pathogens. The molecular identification of the tick samples was based on the amplification of the 12S rRNA mitochondrial Deoxyribonucleic acid. At the same time, those of Rickettsia and Borrelia species were carried out by amplifying fragments of gltA and ompB genes for Rickettsia and flaB gene for Borrelia spp. Thereafter, the positive amplicons for Rickettsia ompB were sequenced and further analyzed. Borrelia PCRs were negative; therefore, sequencing could not be performed. Results: Eight species of ticks belonging to three genera; Rhipicephalus, Amblyomma, and Haemaphysalis, were identified. A total of 27% (320/1,200) samples were confirmed positive for Rickettsia, of which 23% (74/320) were positive for ompB genes. Phylogenetic analysis of ompB revealed a high homology to rickettsial reference strains from GenBank, with no positive result for Borrelia. The generated sequences showed homology with R. africae-KX227790 (100%), R. parkeri-KY113111 (99.8%), R. peacockii (99.3%), and R. slovaca-JX683122 (99.1%) representative sequences in GenBank. Conclusion: The findings from this study revealed that ticks harbored Rickettsia species with possible zoonotic potential.

Hypervirulent Klebsiella pneumoniae in a South African tertiary hospital-Clinical profile, genetic determinants, and virulence in Caenorhabditis elegans.

Introduction: A distinct strain of Klebsiella pneumoniae (K. pneumoniae) referred to as hypervirulent (hvKp) is associated with invasive infections such as pyogenic liver abscess in young and healthy individuals. In South Africa, limited information about the prevalence and virulence of this hvKp strain is available. The aim of this study was to determine the prevalence of hvKp and virulence-associated factors in K. pneumoniae isolates from one of the largest tertiary hospitals in a South African province. Methods: A total of 74 K. pneumoniae isolates were received from Pelonomi Tertiary Hospital National Health Laboratory Service (NHLS), Bloemfontein. Virulence-associated genes (rmpA, capsule serotype K1/K2, iroB and irp2) were screened using Polymerase Chain Reaction (PCR). The iutA (aerobactin transporter) gene was used as a primary biomarker of hvKp. The extracted DNAs were sequenced using the next-generation sequencing pipeline and the curated sequences were used for phylogeny analyses using appropriate bioinformatic tools. The virulence of hvKp vs. classical Klebsiella pneumoniae (cKp) was investigated using the Caenorhabditis elegans nematode model. Results: Nine (12.2%) isolates were identified as hvKp. Moreover, hvKp was significantly (p < 0.05) more virulent in vivo in Caenorhabditis elegans relative to cKp. The virulence-associated genes [rmpA, iroB, hypermucoviscous phenotype (hmv) phenotype and capsule K1/K2] were significantly (p < 0.05) associated with hvKp. A homology search of the curated sequences revealed a high percentage of identity between 99.8 and 100% with other homologous iutA gene sequences of other hvKp in the GenBank. Conclusion: Findings from this study confirm the presence of hvKp in a large tertiary hospital in central South Africa. However, the low prevalence and mild to moderate clinical presentation of infected patients suggest a marginal threat to public health. Further studies in different settings are required to establish the true potential impact of hvKp in developing countries.

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.

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.

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.