Evaluation of serological assays available in a biosafety level 2 laboratory and their application for survey of Middle East respiratory syndrome coronavirus among livestock in Ethiopia.

A serological survey of Middle East respiratory syndrome coronavirus (MERS-CoV) was conducted among dromedary camels and herbivorous animals sharing the same pasturage in Ethiopia. The pseudotyped vesicular stomatitis virus coated with the spike protein of MERS-CoV was used in virus neutralization (VN) tests performed in a biosafety level (BSL)-2 laboratory. The results were similar to those obtained from the VN test using live MERS-CoV and were more sensitive than the ELISA performed using synthetic MERS S1 fragment as the antigen as well as the competitive ELISA performed using a monoclonal antibody against MERS-CoV. According to the comprehensive results of the four types of serodiagnosis methods, positive antibodies were detected only in dromedary camels and the remaining herbivorous animals were not infected with the virus. Moreover, using the present procedure, serological tests for MERS-CoV can be conducted even in BSL 2 laboratory.

Middle East Respiratory Syndrome Coronavirus in Dromedaries in Ethiopia Is Antigenically Different From the Middle East Isolate EMC.

Middle East respiratory syndrome (MERS) is an emerging respiratory disease caused by the MERS coronavirus (MERS-CoV). MERS has been endemic to Saudi Arabia since 2012. The reservoir of MERS-CoV is the dromedary camel, suggesting that MERS is primarily a zoonotic disease. MERS-CoV is common in dromedaries throughout the Middle East, North Africa, and East Africa as evidenced by neutralizing antibodies against MERS-CoV; however, human cases have remained limited to the Middle East. To better understand the cause of this difference, the virological properties of African camel MERS-CoV were analyzed based on the spike (S) protein in Ethiopia. Nasal swabs were collected from 258 young dromedaries (≤ 2 years old) in the Afar region of Ethiopia, of which 39 were positive for MERS-CoV, as confirmed by genetic tests. All positive tests were exclusive to the Amibara woreda region. Using next-generation sequencing, two full-length genomes of Amibara isolates were successfully decoded; both isolates belonged to the C2 clade based on phylogenetic analysis of full-length and S protein sequences. Recombinant EMC isolates of MERS-CoV, in which the S protein is replaced with those of Amibara isolates, were then generated to test the roles of these proteins in viral properties. Amibara S recombinants replicated more slowly in cultured cells than in EMC S recombinants. In neutralizing assays, Amibara S recombinants were neutralized by lower concentrations of sera from both Ethiopian dromedaries and EMC isolate (wild-type)-immunized mouse sera, relative to the EMC S recombinants, indicating that viruses coated in the Amibara S protein were easier to neutralize than the EMC S protein. Neutralization experiments performed using S1/S2 chimeric recombinants of the EMC and Amibara S proteins showed that the neutralization profile was dependent on the S1 region of the S protein. These results suggest that the slower viral replication and the ease of neutralization seen in the Ethiopian MERS-CoV are due to strain-specific differences in the S protein and may account for the absence of human MERS-CoV cases in Ethiopia.

Influenza D Virus Infection in Dromedary Camels, Ethiopia.

Influenza D virus has been found to cause respiratory diseases in livestock. We surveyed healthy dromedary camels in Ethiopia and found a high seroprevalence for this virus, in contrast to animals co-existing with the camels. Our observation implies that dromedary camels may play an important role in the circulation of influenza D virus.

Characterization of novel monoclonal antibodies against the MERS-coronavirus spike protein and their application in species-independent antibody detection by competitive ELISA.

Since discovering the Middle East respiratory syndrome coronavirus (MERS-CoV) as a causative agent of severe respiratory illness in the Middle East in 2012, serological testing has been conducted to assess antibody responses in patients and to investigate the zoonotic reservoir of the virus. Although the virus neutralization test is the gold standard assay for MERS diagnosis and for investigating the zoonotic reservoir, it uses live virus and so must be performed in high containment laboratories. Competitive ELISA (cELISA), in which a labeled monoclonal antibody (MAb) competes with test serum antibodies for target epitopes, may be a suitable alternative because it detects antibodies in a species-independent manner. In this study, novel MAbs against the spike protein of MERS-CoV were produced and characterized. One of these MAbs was used to develop a cELISA. The cELISA detected MERS-CoV-specific antibodies in sera from MERS-CoV-infected rats and rabbits immunized with the spike protein of MERS-CoV. The MAb-based cELISA was validated using sera from Ethiopian dromedary camels. Relative to the neutralization test, the cELISA detected MERS-CoV-specific antibodies in 66 Ethiopian dromedary camels with a sensitivity and specificity of 98% and 100%, respectively. The cELISA and neutralization test results correlated well (Pearson's correlation coefficients=0.71-0.76, depending on the cELISA serum dilution). This cELISA may be useful for MERS epidemiological investigations on MERS-CoV infection.

Serological evidence of hepatitis E virus infection in dromedary camels in Ethiopia.

The genome of dromedary camel hepatitis E virus (DcHEV) has been detected in stool and serum samples from dromedary camels, but the sero-epidemiological information of DcHEV infection remains unclear. A total of 246 serum samples collected from dromedary camels (Camelus dromedarius) in Ethiopia, and 40 serum samples from Bactrian camels (Camelus ferus) in Mongolia were examined for the detection of anti-DcHEV IgG antibody by a newly developed enzyme-linked immunosorbent assay (ELISA) by using DcHEV-like particles (DcHEV-LPs) as the antigen. The results revealed that 55 of the 246 (22.4%) dromedary camels were positive for anti-DcHEV IgG, whereas all 40 samples from the Bactrian camels were negative for DcHEV IgG antibody. A total of 98 serum samples from dromedary camels, including 25 anti-DcHEV-IgG positive samples, were used for the detection of DcHEV RNA by reverse transcription-polymerase chain reaction (RT-PCR), however, no positive samples were identified. These results suggested that the DcHEV infection occurred in the dromedary camels in Ethiopia. Further studies are required to determine whether Bactrian camels are susceptible to DcHEV infection. In addition, not only DcHEV-LPs, but also virus-like particles (VLPs) delivered from G1, G3 and G5 HEV are likely applicable for the detection of the anti-DcHEV IgG antibody.

Serological survey of viral diseases relating to reproductive failure among Artiodactyla in Ethiopian Camelus dromedarius.

Serological surveys were performed on Ethiopian camels with a history of abortion to investigate the presence of antibodies against viruses that infect animals classified in the order Artiodactyla. In 2013, 120 serum samples were collected from camels in various parts of Ethiopia. Several viruses related to abortion in ruminants were prevalent. In particular, antibodies against bluetongue virus, were detected at a high rate (76.7% of samples). Additionally, antibodies against Akabane virus and Japanese encephalitis virus were also detected in samples from more than 40% of the camels; however, their antibody titers were relatively low.

Testicular cytological profiles of apparently healthy male dromedary camels during rutting and non-rutting periods.

The aim of this study was to evaluate testicular cytological profiles of apparently healthy dromedary bulls during rutting and non-rutting periods. Pairs of testes from 26 (18 non-rutting and 8 rutting seasons) dromedary bulls 6-12 years old that were slaughtered at Akaki, Addis Ababa abattoir were sampled. A 21 gauge needle attached to 20mL syringe was used to collect Testicular Fine Needle Aspiration (TFNA) samples and five aspiration smears were prepared from each testis. A total of 312 slides (260 Testicular fine Needle Aspiration and 52 imprints) were examined. The mod ified May-Grunwald Giemsa (mMGG) technique and a light microscope were used to assess cellularity, morphology and quantification of the testicular. Sertoli and spermatogenic cells were identified and counted. The spermatic index (SI), Sertoli cell index (SEI) and the relationship between SI and SEI indexes (SSEI) were used to assess the ratio between mature spermatozoa and nursing cells. There were differences (P<0.05) between the rutting and non-rutting seasons among the spermatogenic and Sertoli cells. There were no differences between groups for primary spermatocyte numbers, early spermatid numbers and SSEI. There was no differences (P>0.05) between TFNA and imprint smear slides of the testicular cells except for Sertoli cell count and SEI. Filarial worm larvae were present on the TFNA smear slides of four animals. Imprint and TFNA smear slides had comparable cytological profiles in dromedary bulls and significant differences were observed between rutting and non-rutting periods.

Camelus dromedarius brucellosis and its public health associated risks in the Afar National Regional State in northeastern Ethiopia.

BACKGROUND: A cross-sectional study was carried out in four districts of the Afar region in Ethiopia to determine the prevalence of brucellosis in camels, and to identify risky practices that would facilitate the transmission of zoonoses to humans. This study involved testing 461 camels and interviewing 120 livestock owners. The modified Rose Bengal plate test (mRBPT) and complement fixation test (CFT) were used as screening and confirmatory tests, respectively. SPSS 16 was used to analyze the overall prevalence and potential risk factors for seropositivity, using a multivariable logistic regression analysis. RESULTS: In the camel herds tested, 5.4% had antibodies against Brucella species, and the district level seroprevalence ranged from 11.7% to 15.5% in camels. The logistic regression model for camels in a herd size > 20 animals (OR = 2.8; 95% CI: 1.16-6.62) and greater than four years of age (OR = 4.9; 95% CI: 1.45-16.82) showed a higher risk of infection when compared to small herds and those ≤ 4 years old. The questionnaire survey revealed that most respondents did not know about the transmission of zoonotic diseases, and that their practices could potentially facilitate the transmission of zoonotic pathogens. CONCLUSIONS: The results of this study revealed that camel brucellosis is prevalent in the study areas. Therefore, there is a need for implementing control measures and increasing public awareness in the prevention methods of brucellosis.