Molecular phylogeny of coronaviruses and host receptors among domestic and close-contact animals reveals subgenome-level conservation, crossover, and divergence.

BACKGROUND: Coronaviruses have the potential to cross species barriers. To learn the molecular intersections among the most common coronaviruses of domestic and close-contact animals, we analyzed representative coronavirus genera infecting mouse, rat, rabbit, dog, cat, cattle, white-tailed deer, swine, ferret, mink, alpaca, Rhinolophus bat, dolphin, whale, chicken, duck and turkey hosts; reference or complete genome sequences were available for most of these coronavirus genera. Protein sequence alignments and phylogenetic trees were built for the spike (S), envelope (E), membrane (M) and nucleocapsid (N) proteins. The host receptors and enzymes aminopeptidase N (APN), angiotensin converting enzyme 2 (ACE2), sialic acid synthase (SAS), transmembrane serine protease 2 (TMPRSS2), dipeptidyl peptidase 4 (DPP4), cathepsin L (and its analogs) and furin were also compared. RESULTS: Overall, the S, E, M, and N proteins segregated according to their viral genera (α, ß, or γ), but the S proteins of alphacoronaviruses lacked conservation of phylogeny. Interestingly, the unique polybasic furin cleavage motif found in severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) but not in severe acute respiratory syndrome coronavirus (SARS-CoV) or Middle East respiratory syndrome coronavirus (MERS-CoV) exists in several ß-coronaviruses and a few α- or γ-coronaviruses. Receptors and enzymes retained host species-dependent relationships with one another. Among the hosts, critical ACE2 residues essential for SARS-CoV-2 spike protein binding were most conserved in white-tailed deer and cattle. CONCLUSION: The polybasic furin cleavage motif found in several ß- and other coronaviruses of animals points to the existence of an intermediate host for SARS-CoV-2, and it also offers a counternarrative to the theory of a laboratory-engineered virus. Generally, the S proteins of coronaviruses show crossovers of phylogenies indicative of recombination events. Additionally, the consistency in the segregation of viral proteins of the MERS-like coronavirus (NC_034440.1) from pipistrelle bat supports its classification as a ß-coronavirus. Finally, similarities in host enzymes and receptors did not always explain natural cross-infections. More studies are therefore needed to identify factors that determine the cross-species infectivity of coronaviruses.

Mechanisms of ivermectin-induced wound healing.

BACKGROUND: Wounds cause structural and functional discontinuity of an organ. Wound healing, therefore, seeks to re-establish the normal morphology and functionality through intertwined stages of hemostasis, inflammation, proliferation, and tissue remodelling. Ivermectin, a macrolide, has been used as an endectoparasiticide in human and veterinary medicine practice for decades. Here, we show that ivermectin exhibits wounding healing activity by mechanisms independent of its well-known antiparasitic activity. This study aimed to evaluate the wound healing property of ivermectin cream using histochemistry and enzyme-linked immunosorbent assay techniques. RESULTS: Non-irritant dose of ivermectin cream (0.03-1%) decreased wound macroscopic indices such as exudation, edge edema, hyperemia, and granulation tissue deposition by day 9 compared to day 13 for the vehicle-treated group. This corresponded with a statistically significant wound contraction rate, hydroxyproline deposition, and a decreased time to heal rate. The levels of growth factors TGF-ß1 and VEGF were significantly elevated on day 7 but decreased on day 21. This corresponded with changes in cytokines (IL-1α, IL-4, IL-10, and TNF-α) and eicosanoids (LTB4, PGE2, and PGD2) levels on days 7 and 21.. Interestingly, low doses of ivermectin cream (0.03-0.1%) induced wound healing with minimal scarring compared to higher doses of the cream and the positive control, Silver Sulfadiazine. CONCLUSION: Ivermectin promotes wound healing partly through modulation of the inflammatory process and the levels of Transforming Growth Factor-Beta 1 and Vascular Endothelial Growth Factor. Low doses of ivermectin cream have the potential to be used in treating wounds with minimal scar tissue formation.

Molecular-based cross-species evaluation of bovine coronavirus infection in cattle, sheep and goats in Ghana.

BACKGROUND: Apart from the huge worldwide economic losses often occasioned by bovine coronavirus (BCoV) to the livestock industry, particularly with respect to cattle rearing, continuous surveillance of the virus in cattle and small ruminants is essential in monitoring variations in the virus that could enhance host switching. In this study, we collected rectal swabs from a total of 1,498 cattle, sheep and goats. BCoV detection was based on reverse transcriptase polymerase chain reaction. Sanger sequencing of the partial RNA-dependent RNA polymerase (RdRp) region for postive samples were done and nucleotide sequences were compared with homologous sequences from the GenBank. RESULTS: The study reports a BCoV prevalence of 0.3%, consisting of 4 positive cases; 3 goats and 1 cattle. Less than 10% of all the animals sampled showed clinical signs such as diarrhea and respiratory distress except for high temperature which occurred in > 1000 of the animals. However, none of the 4 BCoV positive animals manifested any clinical signs of the infection at the time of sample collection. Bayesian majority-rule cladogram comparing partial and full length BCoV RdRp genes obtained in the study to data from the GenBank revealed that the sequences obtained from this study formed one large monophyletic group with those from different species and countries. The goat sequences were similar to each other and clustered within the same clade. No major variations were thus observed between our isolates and those from elsewhere. CONCLUSIONS: Given that Ghana predominantly practices the extensive and semi-intensive systems of animal rearing, our study highlights the potential for spillover of BCoV to small ruminants in settings with mixed husbandry and limited separation between species.

Serological evidence of Coxiella burnetii infection in slaughtered sheep and goats at Kumasi Abattoir, Ghana.

Q fever, caused by Coxiella burnetii, is an important zoonosis worldwide. Q fever is documented in many parts of the world; however, information on the disease in Ghana is scanty. This study was therefore conducted to provide evidence of exposure of sheep and goats slaughtered at the Kumasi Abattoir to Coxiella burnetii. A total of 350 serum samples collected from 175 sheep and 175 goats were analyzed for the presence of C. burnetii antibodies using a commercial ELISA kit (ID Vet). Results of the study established a seroprevalence of 28.57% in goats, 16.57% in sheep and an overall seroprevalence of 22.29% in sheep and goats; 20.57% for male sheep, 23.86% for female sheep, 26.44% for male goats and 30.68% for female goats. Results showed that goats are more at risk to the infection than sheep however sex is not a risk factor. This study confirms the existence of Q fever in sheep and goats in Ghana hence, the disease should be considered as a public health risk to workers at the abattoir and other stakeholders in the sheep and goat production chain.

Histological and immunohistochemical assessments of pneumonia in sheep slaughtered at Ibadan, Nigeria and Kumasi, Ghana.

There is a decline in sheep production and contribution to the economy. This study described the histological pattern, immunohistochemically demonstrate bacterial and viral agents of pneumonia in sheep slaughtered in metropolitan abattoirs in Nigeria and Ghana. A total of 805 (600 in Ghana and 205 in Nigeria) sheep lungs were examined for pneumonic lesions. Sections were fixed in 10% formalin for histological and immunohistochemical analysis. Pneumonia of 8.7% was in Ghana and 13.3% in Nigeria. The histological findings included bronchopneumonia (16/70), broncho-interstitial pneumonia (9/70), interstitial pneumonia (9/70) and proliferative pneumonia (7/70). Ten (14.3%) cases were positive for PPRV, PI3V, and RSV antigens while five (7.2%) were positive for multiple viral antigens; including two (PI3V and PPRV), two (RSV and PPRV), and one (PI3V and RSV). RSV was in interstitial pneumonia, PPRV and PI3V were in interstitial and broncho-interstitial pneumonia. PPRV and PI3V antigens 3 (4.4%) were in proliferative alveolitis. Mannheimia haemolytica (2) and Pasteurella multocida (2) infection were in combination with PPRV, RSV, and PI3V. Three sheep lungs (4.4%) were negative for viruses and bacteria. This study revealed ovine pneumonia is still a problem in West Africa, and multivalent vaccine incorporating some of these agents will be useful for the control.

Seroprevalence of Toxoplasma Gondii antibodies in sheep and goats slaughtered at the Kumasi Abattoir, Ghana.

Toxoplasmosis, caused by T. gondii, is an important zoonosis worldwide. In Ghana, information on the disease in humans abounds but scanty in animals. This study was therefore conducted to estimate the seroprevalence of T. gondii infection sheep and goats sampled from the Kumasi Abattoir in Ashanti Region, Ghana. A total of 347 serum samples collected from 170 sheep and 177 goats were analyzed for the presence of T. gondii antibodies using a commercial ELISA kit. Results of this study estimated the seroprevalence of 23.7% in goats an, 35.9% in sheep. In sheep, 24 (35.82%) out of a total of 67 male samples were positive and 37 (35.92%) out of a total of 104 female samples were positive while in goats, 6 (8.2%) bucks out of a total of 73 were positive while 36 (34.6%) does out of a total of 104 were positive. There was a significant difference in the rate of seropositivity of female goats (p-value 0.01). This study confirms the existence of T. gondii infection in small ruminants in Ghana and it showed that sheep and dogs are more at risk to T. gondii infection hence meat from such animals could be a potential risk to public health if consumed raw or undercooked.

Rabies virus in slaughtered dogs for meat consumption in Ghana: A potential risk for rabies transmission.

Dog-mediated rabies is responsible for approximately 60,000 human deaths annually worldwide. Although dog slaughter for human consumption and its potential risk for rabies transmission has been reported, mainly in some parts of Western Africa and South-East Asia, more information on this and factors that influence dog meat consumption is required for a better understanding from places like Ghana where the practice is common. We tested 144 brain tissues from apparently healthy dogs slaughtered for human consumption for the presence of rabies viruses using a Lyssavirus-specific real-Time RT-PCR. Positive samples were confirmed by virus genome sequencing. We also administered questionnaires to 541 dog owners from three regions in Ghana and evaluated factors that could influence dog meat consumption. We interacted with butchers and observed slaughtering and meat preparation procedures. Three out of 144 (2.1%) brain tissues from apparently healthy dogs tested positive for rabies virus RNA. Two of the viruses with complete genomes were distinct from one another, but both belonged to the Africa 2 lineage. The third virus with a partial genome fragment had high sequence identity to the other two and also belonged to the Africa 2 lineage. Almost half of the study participants practiced dog consumption [49% (265/541)]. Males were almost twice (cOR = 1.72, 95% CI (1.17-2.52), p-value = .006) as likely to consume dog meat compared to females. Likewise, the Frafra tribe from northern Ghana [cOR = 825.1, 95% CI (185.3-3672.9), p-value < .0001] and those with non-specific tribes [cOR = 47.05, 95% CI (10.18-217.41), p-value < .0001] presented with higher odds of dog consumption compared to Ewes. The butchers used bare hands in meat preparation. This study demonstrates the presence of rabies virus RNA in apparently healthy dogs slaughtered for human consumption in Ghana and suggests a potential risk for rabies transmission. Veterinary departments and local assemblies are recommended to monitor and regulate this practice.

Sero-molecular epidemiology of hepatitis E virus in pigs and human contacts in Ghana.

BACKGROUND: Hepatitis E virus (HEV) is among the leading causes of viral hepatitis in most developing countries. Zoonotic acquisition of HEV genotype 3 from swine has come into focus more recently. Available studies on HEV in Ghana and other countries in the region do not provide enough information towards understanding the epidemiology of HEV in human and animal populations. Towards this end, we conducted a comparative cross-sectional study to determine the seroprevalence and risk factors associated with HEV exposure, both in swine and humans working on pig farms in typical local settings. The presence of viral RNA in human and swine samples was also evaluated, along with classification of viral sequences from HEV-positive samples. METHODS: Structured questionnaires soliciting information on pigs reared, as well as socio-demographic information including age, sex and educational background of humans was collected. A total of 10 ml and 5 ml of whole blood was collected from pigs and human participants respectively. ELISA and real-time RT-PCR were performed on the sera for the qualitative detection of IgG antibodies to hepatitis E virus and viral RNA, respectively. RESULTS: Five hundred and forty-four (544) human participants including 264 swine contacts and 280 swine non-contacts were enrolled in the study. Although the proportion of HEV IgG antibodies was higher in contact groups (114; 54.3%) than non-contact groups (96; 45.7%), a multivariate analysis did not show any significant difference. No HEV RNA was detected in human samples. Similarly, 720 pigs were sampled from 18 farms located in five regions in Ghana. Twenty-three (23) of the pigs (3.2, 95%CI = 2.0-4.8) were positive for HEV RNA by real-time RT-PCR testing. Sequences obtained from HEV-positive samples were found to share high sequence identities with each other and clustered with other genotype 3 viruses indicating the existence of circulating zoonotic genotype 3 viruses on farms. Although we did not find evidence of pig to human transmission of HEV genotype 3, the presence of this genotype in pigs shows the potential for possible zoonotic transmission in African farm settings and buttresses the importance of active surveillance for the infection among at risk populations.

Detection and genomic characterization of hepatitis E virus genotype 3 from pigs in Ghana, Africa.

BACKGROUND: Hepatitis E virus (HEV) is a major cause of human hepatitis worldwide. Zoonotic genotypes of the virus have been found in diverse animal species with pigs playing a major role. Putative risk of zoonotic infection from livestock particularly swine in Sub-Saharan Africa including Ghana is poorly understood due to scarcity of available data, especially HEV sequence information. METHODS: Serum samples were collected from cattle, sheep, goats and pigs from Kumasi in the Ashanti region of Ghana. Samples were subjected to nested RT-PCR screening and quantification of HEV RNA-positive samples using real-time RT-PCR and the World Health Organization International Standard for HEV. Testing of all pig samples for antibodies was done by ELISA. Sanger sequencing and genotyping was performed and one representative complete genome was generated to facilitate genome-wide comparison to other available African HEV sequences by phylogenetic analysis. RESULTS: A total of 420 samples were available from cattle (n = 105), goats (n = 124), pigs (n = 89) and sheep (n = 102). HEV Viral RNA was detected only in pig samples (10.1%). The antibody detection rate in pigs was 77.5%, with positive samples from all sampling sites. Average viral load was 1 × 105 (range 1.02 × 103 to 3.17 × 105) International Units per mL of serum with no statistically significant differences between age groups (≤ 6 month, > 6 months) by a T-test comparison of means (t = 1.4272, df = 7, p = 0.1966). Sequences obtained in this study form a monophyletic group within HEV genotype 3. Sequences from Cameroon, Ghana, Burkina Faso and Madagascar were found to share a most recent common ancestor; however this was not the case for other African HEV sequences. CONCLUSION: HEV genotype 3 is highly endemic in pigs in Ghana and likely poses a zoonotic risk to people exposed to pigs. HEV genotype 3 in Ghana shares a common origin with other virus strains from Sub-Saharan Africa.

Sero-prevalence, cross-species infection and serological determinants of prevalence of Bovine Coronavirus in Cattle, Sheep and Goats in Ghana.

Cattle, goats and sheep are dominant livestock species in sub-Saharan Africa, with sometimes limited information on the prevalence of major infectious diseases. Restrictions due to notifiable epizootics complicate the exchange of samples in surveillance studies and suggest that laboratory capacities should be established domestically. Bovine Coronavirus (BCoV) causes mainly enteric disease in cattle. Spillover to small ruminants is possible. Here we established BCoV serology based on a recombinant immunofluorescence assay for cattle, goats and sheep, and studied the seroprevalence of BCoV in these species in four different locations in the Greater Accra, Volta, Upper East, and Northern provinces of Ghana. The whole sampling and testing was organized and conducted by a veterinary school in Kumasi, Ashanti Region of Ghana. Among sampled sheep (n = 102), goats (n = 66), and cattle (n = 1495), the seroprevalence rates were 25.8 %, 43.1 % and 55.8 %. For cattle, seroprevalence was significantly higher on larger farms (82.2 % vs 17.8 %, comparing farms with >50 or <50 animals; p = 0.027). Highest prevalence was seen in the Northern province with dry climate, but no significant trend following the north-south gradient of sampling sites was detected. Our study identifies a considerable seroprevalence for BCoV in Ghana and provides further support for the spillover of BCoV to small ruminants in settings with mixed husbandry and limited separation between species.

Syndromic survey and molecular analysis of influenza viruses at the human-swine interface in two West African cosmopolitan cities suggest the possibility of bidirectional interspecies transmission.

Influenza viruses are frequently transmitted between pigs and their handlers, and among pig handlers. However, reports on socio-environmental variables as potential risk factors associated with transmission of influenza in West African swine production facilities are very scarce. Syndromic survey for influenza was therefore conducted in Ibadan, Nigeria, and Kumasi, Ghana, in order to identify and elucidate selected socio-environmental variables that may contribute to the occurrence and distribution of influenza-like illness (ILI) among swine industry workers. In addition, molecular analyses were conducted to elucidate the nature of influenza viruses circulating at the human-swine interface in these cities and better understand the dynamics of their transmission. Influenza viruses were detected by type-specific and subtype-specific RT-PCR. Sequencing and phylogenetic analyses were carried out. Socio-environmental variables were tested by both univariable and multivariable regression methods for significance at p < 0.05. Three risk factors for ILI were identified in each city. These included "frequency of visit of pig handler to pig pen or lairage" (Ibadan: risk ratio [RR] = 1.54, 95% confidence interval [CI] = 1.36-1.79, p = 0.02; Kumasi: RR = 1.28, 95% CI = 1.11-1.71, p = 0.01) and "pig handler's awareness about biosecurity measures" (Ibadan: RR = 7.09, 95% CI = 2.36-21.32, p < 0.001; Kumasi: RR = 4.84, 95% CI = 1.98-11.80, p < 0.001). Influenza A(H1N1)pdm09 viruses, with M genes closely related to those which circulated among pigs in the two cities during the same period, were detected among Nigerian and Ghanaian pig industry workers. These findings suggest the possibility of bidirectional transmission of influenza at the human-swine interface in these cities and underscore the need for more extensive molecular studies. Risk factors identified may assist in the control of human-to-human and human-to-swine transmission of influenza in the West African swine industry.

Potential Intermediate Hosts for Coronavirus Transmission: No Evidence of Clade 2c Coronaviruses in Domestic Livestock from Ghana.

The emergence of Middle East Respiratory Syndrome Coronavirus (MERS-CoV), nearly a decade ago with worldwide distribution, was believed to be of zoonotic origin from bats with dromedary camels as intermediate hosts. There is a likelihood of other domestic livestock serving as intermediate hosts for this virus. The presence of coronaviruses, closely related to MERS-CoV in Ghanaian bats, presented the opportunity to test the hypothesis of transmissibility of this virus through domestic livestock species. The possible interactions between livestock and bats in 31 household farms were accessed by observation and interviews with farmers. Rectal swabs and serum from cattle, sheep, goats, donkeys, and swine from commercial and household farms were tested for MERS-CoV and a Nycteris sp. bat coronavirus, previously detected in Ghana. A pan-PCR assay to detect clade 2c viruses and recombinant immunofluorescence assay to detect anti-spike IgG antibodies against the target viruses were used. Likely contact between livestock and bats was determined for 13 farms (41.9%) that reported confining their livestock and also observing bats in their homes. Livestock were left unconfined on eight farms (25.8%) that also observed bats roosting in trees close to their homes. No viral RNA or antibodies against the two coronaviruses were detected in any of the livestock species tested. Cattle, sheep, goats, donkeys, and swine are not likely hosts of clade 2c coronaviruses.

Development of a Whole-Virus ELISA for Serological Evaluation of Domestic Livestock as Possible Hosts of Human Coronavirus NL63.

Known human coronaviruses are believed to have originated in animals and made use of intermediate hosts for transmission to humans. The intermediate hosts of most of the human coronaviruses are known, but not for HCoV-NL63. This study aims to assess the possible role of some major domestic livestock species as intermediate hosts of HCoV-NL63. We developed a testing algorithm for high throughput screening of livestock sera with ELISA and confirmation with recombinant immunofluorescence assay testing for antibodies against HCoV-NL63 in livestock. Optimization of the ELISA showed a capability of the assay to significantly distinguish HCoV-NL63 from HCoV-229E (U = 27.50, p < 0.001) and HCoV-OC43 (U = 55.50, p < 0.001) in coronavirus-characterized sera. Evaluation of the assay with collected human samples showed no significant difference in mean optical density values of immunofluorescence-classified HCoV-NL63-positive and HCoV-NL63-negative samples (F (1, 215) = 0.437, p = 0.509). All the top 5% (n = 8) most reactive human samples tested by ELISA were HCoV-NL63 positive by immunofluorescence testing. In comparison, only a proportion (84%, n = 42) of the top 25% were positive by immunofluorescence testing, indicating an increased probability of the highly ELISA reactive samples testing positive by the immunofluorescence assay. None of the top 5% most ELISA reactive livestock samples were positive for HCoV-NL63-related viruses by immunofluorescence confirmation. Ghanaian domestic livestock are not likely intermediate hosts of HCoV-NL63-related coronaviruses.

THE PREVALENCE OF TUBERCULOSIS IN CATTLE AND THEIR HANDLERS IN NORTH TONGU, VOLTA REGION, GHANA.

BACKGROUND: The need to understand the contribution of bovine tuberculosis (BTB) to the general tuberculosis burden in a poor resource setting is paramount. The aim of this study is to determine the burden of BTB among herdsmen and cattle in the North Tongu district of Volta Region in Ghana. MATERIALS AND METHODS: A cross- sectional study was conducted in the North Tongu District of the Volta Region between the period of October 2011- March 2012. A well-structured questionnaire was used to collect socio-demographic information and possible risk factor information on cattle from participants. Sputum samples from 68 herdsmen and blood samples from 200 cattle belonging to these herdsmen were also collected. Sputum samples were analyzed using Ziehl- Neelsen staining while Anigen Rapid BTB Test was used for Cattle blood samples. RESULTS: Ninety percent (61/68) of respondents were also found to consume fresh milk while 84% (57/68) do not use protective clothing. Of a total of 1580 cattle owned by the herdsmen, 200 cattle consisting of 14 bulls and 186 cows were screened where the prevalence of bovine TB was 19% (38/200) and those affected were all females. All (100%) human sample tested negative for Acid-Fast Bacilli (AFB). However, the seropositivity of cattle and kraal density were statistically associated (p= 0.001). CONCLUSION: Bovine TB is prevalent in cattle in North Tongu district. Although herdsmen indulge in risky lifestyles that expose them to BTB, a zero prevalence of BTB was observed, further study is envisaged using a larger sample size.