A novel multiplex qPCR­HRM assay for the simultaneous detection of four abortive zoonotic agents in cattle, sheep, and goats.

Abortifacient pathogens induce substantial economic losses in the livestock industry worldwide, and many of these pathogens are zoonotic, impacting human health. As Brucella spp., Coxiella burnetii, Leptospira spp., and Listeria monocytogenes cause abortion, rapid differential molecular diagnostic tests are needed to facilitate early and accurate detection of abortion to establish effective control measures. However, the available molecular methods are laborious, time-consuming, or costly. Therefore, we developed and validated a novel multiplex real-time polymerase chain reaction (qPCR) method based on high-resolution melting (HRM) curve analysis to simultaneously detect and differentiate four zoonotic abortifacient agents in cattle, goats, and sheep. Our HRM assay generated four well-separated melting peaks allowing the differentiation between the four zoonotic abortifacients. Out of 216 DNA samples tested, Brucella spp. was detected in 45 samples, Coxiella burnetii in 57 samples, Leptospira spp. in 12 samples, and Listeria monocytogenes in 19 samples, co-infection with Brucella spp. and Coxiella burnetii in 41 samples, and 42 samples were negative. This assay demonstrated good analytical sensitivity, specificity, and reproducibility. This is a valuable rapid, cost-saving, and reliable diagnostic tool for detecting individual and co-infections for zoonotic abortifacient agents in ruminants.

Emergence of High Pathogenicity Avian Influenza Virus H5N1 Clade 2.3.4.4b in Wild Birds and Poultry in Botswana.

Numerous outbreaks of high-pathogenicity avian influenza (HPAI) were reported during 2020-2021. In Africa, H5Nx has been detected in Benin, Burkina Faso, Nigeria, Senegal, Lesotho, Namibia and South Africa in both wild birds and poultry. Botswana reported its first outbreak of HPAI to the World Organisation for Animal Health (WOAH) in 2021. An H5N1 virus was detected in a fish eagle, doves, and chickens. Full genome sequence analysis revealed that the virus belonged to clade 2.3.4.4b and showed high identity within haemagglutinin (HA) and neuraminidase proteins (NA) for viruses identified across a geographically broad range of locations. The detection of H5N1 in Botswana has important implications for disease management, wild bird conservation, tourism, public health, economic empowerment of vulnerable communities and food security in the region.

First molecular characterization of poxviruses in cattle, sheep, and goats in Botswana.

BACKGROUND: Poxviruses within the Capripoxvirus, Orthopoxvirus, and Parapoxvirus genera can infect livestock, with the two former having zoonotic importance. In addition, they induce similar clinical symptoms in common host species, creating a challenge for diagnosis. Although endemic in the country, poxvirus infections of small ruminants and cattle have received little attention in Botswana, with no prior use of molecular tools to diagnose and characterize the pathogens. METHODS: A high-resolution melting (HRM) assay was used to detect and differentiate poxviruses in skin biopsy and skin scab samples from four cattle, one sheep, and one goat. Molecular characterization of capripoxviruses and parapoxviruses was undertaken by sequence analysis of RPO30 and GPCR genes. RESULTS: The HRM assay revealed lumpy skin disease virus (LSDV) in three cattle samples, pseudocowpox virus (PCPV) in one cattle sample, and orf virus (ORFV) in one goat and one sheep sample. The phylogenetic analyses, based on the RPO30 and GPCR multiple sequence alignments showed that the LSDV sequences of Botswana were similar to common LSDV field isolates encountered in Africa, Asia, and Europe. The Botswana PCPV presented unique features and clustered between camel and cattle PCPV isolates. The Botswana ORFV sequence isolated from goat differed from the ORFV sequence isolated from sheep. CONCLUSIONS: This study is the first report on the genetic characterization of poxvirus diseases circulating in cattle, goats, and sheep in Botswana. It shows the importance of molecular methods to differentially diagnose poxvirus diseases of ruminants.

Evaluation of the sensitivity and specificity of the lateral flow assay, Rose Bengal test and the complement fixation test for the diagnosis of brucellosis in cattle using Bayesian latent class analysis.

This study was conducted to evaluate the sensitivity (Se) and specificity (Sp) of the Rose Bengal test (RBT), complement fixation test (CFT), the serum lateral flow assay (LFAserum) and the blood lateral flow assay (LFAblood) for the detection of antibodies to Brucella spp. using Bayesian latent class models (BLCMs). Sera and whole blood were collected from naturally infected cattle reared in smallholder, small-scale commercial and large-scale commercial farms in Zimbabwe (n = 1022) and Botswana (n = 770). The BLCMs were fitted under the assumption that conditional dependences existed between the tests. Based on the conditional dependence model, the RBT had the highest Se of 0.897 (95 % Probability Intervals: 0.854; 0.932) compared to 0.827 (0.773; 0.872), 0.812 (0.76; 0.858) and 0.809 (0.785; 0.832) for the LFAserum, LFAblood and CFT, respectively. The CFT recorded a higher Sp of 0.999 (0.995; 1.000) than the LFAserum 0.996 (0.99; 1.000), the LFAblood 0.984 (0.976; 0.991) and the RBT 0.969 (0.959; 0.978). The data indicated that both the Se and Sp of RBT and CFT and the Sp of LFAserum and LFAblood were conditionally independent, while the Se appeared to be conditionally dependent. These results indicated that none of the evaluated tests had perfect Se and Sp and consequently could not be used alone for the diagnosis of brucellosis in cattle from the studied farming sectors. Thus, based on high Se and Sp, respectively, a brucellosis testing regimen using the RBT (screening) and the LFA (confirmatory) may be considered.

First molecular characterization of avian paramyxovirus-1 (Newcastle disease virus) in Botswana.

Avian paramyxovirus-1 (APMV-1), the causative agent of Newcastle disease (ND) in domestic and wild avian species, has recently been reported and characterized in five southern African countries (i.e. Mozambique, Namibia, South Africa, Zambia and Zimbabwe). Since APMV-1s have never been characterized in Botswana, this study was undertaken to determine the genotype circulating in the country. Fourteen samples were collected from ND outbreaks in poultry in 2014, 2018 and 2019 and the complete fusion protein gene was sequenced. Phylogenetic analysis revealed that all of the viruses from Botswana clustered in genotype VII.2 (previously subgenotype VIIh) and that they were more related to viruses from South Africa and Mozambique than the other southern African countries (i.e. Namibia, Zambia and Zimbabwe).