Virus-specific antibody secreting cells reside in the peritoneal cavity and systemic immune sites of Atlantic salmon (Salmo salar) challenged intraperitoneally with salmonid alphavirus.

The development and persistence of antibody secreting cells (ASC) after antigenic challenge remain inadequately understood in teleosts. In this study, intraperitoneal (ip) injection of Atlantic salmon (Salmo salar) with salmonid alphavirus (WtSAV3) increased the total ASC response, peaking 3-6 weeks post injection (wpi) locally in the peritoneal cavity (PerC) and in systemic lymphoid tissues, while at 13 wpi the response was only elevated in PerC. At the same time point a specific ASC response was induced by WtSAV3 in PerC and systemic tissues, with the highest frequency in PerC, suggesting a local role. Inactivated SAV (InSAV1) induced comparatively lower ASC responses in all sites, and specific serum antibodies were only induced by WtSAV3 and not by InSAV1. An InSAV1 boost did not increase these responses. Expression of immune marker genes implies a role for PerC adipose tissue in the PerC immune response. Overall, the study suggests the Atlantic salmon PerC as a secondary immune site and an ASC survival niche.

There and back again? A B cell's tale on responses and spatial distribution in teleosts.

Teleost B cells are of special interest due to their evolutionary position and involvement in vaccine-induced adaptive immune responses. While recent progress has revealed uneven distribution of B cell subsets across the various immune sites and that B cells are one of the early responders to infection, substantial knowledge gaps persist regarding their immunophenotypic profile, functional mechanisms, and what factors lead them to occupy different immune niches. This review aims to assess the current understanding of B cell diversity, their spatial distribution in various systemic and peripheral immune sites, how B cell responses initiate, the sites where these responses develop, their trafficking, and the locations where long-term B cell responses take place.

The importance of the Atlantic salmon peritoneal cavity B cell response: Local IgM secreting cells are predominant upon Piscirickettsia salmonis infection.

The intraperitoneal route is favored for administration of inactivated and attenuated vaccines in Atlantic salmon. Nevertheless, the immune responses in the teleost peritoneal cavity (PerC) are still incompletely defined. In this study, we investigated the B cell responses after intraperitoneal Piscirickettsia salmonis (P. salmonis) challenge of Atlantic salmon, focusing on the local PerC response versus responses in the lymphatic organs: spleen and head kidney. We observed a major increase of leukocytes, total IgM antibody secreting cells (ASC), and P. salmonis-specific ASC in the PerC at 3- and 6-weeks post infection (wpi). The increase in ASC frequency was more prominent in the spleen and PerC compared to the head kidney during the observed 6 wpi. The serum antibody response included P. salmonis-specific antibodies and non-specific antibodies recognizing the non-related bacterial pathogen Yersinia ruckeri and the model antigen TNP-KLH. Finally, we present evidence that supports a putative role for the adipose tissue in the PerC immune response.

Salmonid Alphavirus Subtype 3 Induces Prolonged Local B Cell Responses in Atlantic Salmon (Salmo salar) After Intraperitoneal Infection.

B cell responses are a crucial part of the adaptive immune response to viral infection. Infection by salmonid alphavirus subtype 3 (SAV3) causes pancreas disease (PD) in Atlantic salmon (Salmo salar) and is a serious concern to the aquaculture industry. In this study, we have used intraperitoneal (IP) infection with SAV3 as a model to characterize local B cell responses in the peritoneal cavity (PerC) and systemic immune tissues (head kidney/spleen). Intraperitoneal administration of vaccines is common in Atlantic salmon and understanding more about the local PerC B cell response is fundamental. Intraperitoneal SAV3 infection clearly induced PerC B cell responses as assessed by increased frequency of IgM+ B cells and total IgM secreting cells (ASC). These PerC responses were prolonged up to nine weeks post-infection and positively correlated to the anti-SAV3 E2 and to neutralizing antibody responses in serum. For the systemic immune sites, virus-induced changes in B cell responses were more modest or decreased compared to controls in the same period. Collectively, data reported herein indicated that PerC could serve as a peripheral immunological site by providing a niche for prolonged maintenance of the ASC response in Atlantic salmon.

Author Correction: Profiling Atlantic salmon B cell populations: CpG-mediated TLR-ligation enhances IgM secretion and modulates immune gene expression.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Profiling Atlantic salmon B cell populations: CpG-mediated TLR-ligation enhances IgM secretion and modulates immune gene expression.

While TLR-activated pathways are key regulators of B cell responses in mammals, their impact on teleost B cells are scarcely addressed. Here, the potential of Atlantic salmon B cells to respond to TLR ligands was shown by demonstrating a constitutive expression of nucleic-acid sensing TLRs in magnetic sorted IgM+ cells. Of the two receptors recognizing CpG in teleosts, tlr9 was the dominating receptor with over ten-fold higher expression than tlr21. Upon CpG-stimulation, IgM secretion increased for head kidney (HK) and splenic IgM+ cells, while blood B cells were marginally affected. The results suggest that CpG directly affects salmon B cells to differentiate into antibody secreting cells (ASCs). IgM secretion was also detected in the non-treated controls, again with the highest levels in the HK derived population, signifying that persisting ASCs are present in this tissue. In all tissues, the IgM+ cells expressed high MHCII levels, suggesting antigen-presenting functions. Upon CpG-treatment the co-stimulatory molecules cd83 and cd40 were upregulated, while cd86 was down-regulated under the same conditions. Finally, ifna1 was upregulated upon CpG-stimulation in all tissues, while a restricted upregulation was evident for ifnb, proposing that salmon IgM+ B cells exhibit a type I IFN-response.

Investigation of Marek's disease virus from chickens in central Ethiopia.

Marek's disease (MD) is a lymphoproliferative and neuropathic disease of domestic chickens and less commonly, turkeys and quails, caused by a highly contagious, cell-associated, oncogenic herpesvirus. In Ethiopia, MD is believed to be introduced with importation of exotic and crossbred to improve the poultry production and has been reported to be a potential threat to the poultry sector both in backyard and commercial farming systems. This study was aimed at isolation and molecular analysis of MD virus isolates circulating in chicken population in the central part of Ethiopia where commercial farms are populated. From September 2013 to January 2014, clinical and post-mortem examination were conducted on diseased chickens suspected of MD virus infection. Representative spleen and feather follicle samples were collected following sterile procedure, and infectious virus isolation was performed using primary chicken fibroblast cell culture. Cell culture inoculated with suspension of pathological samples developed characteristic MD virus cytopathic effect of rounding of the cells and small plaques. Further analysis of the virus was conducted by conventional PCR amplifying the ICP4 gene fragment from eleven tissue samples using MD virus specific primers. PCR products were further sequenced and analyzed. Nucleotide sequence similarity search of the local isolates resulted a high degree of sequence similarity with Gallid Herpes virus type 2 strain (Marek's disease virus type 1, JN034558). To our knowledge, the present study is the first report conducted on virus isolation and molecular characterization of MD virus isolates circulated in Ethiopia. Eleven ICP4-like gene fragment (318 bp) sequences generated in the present study were uploaded in the public database (KU842366-76). Further research on virus isolation, genetic characterization, and infection dynamics is recommended targeting chickens of all age groups reared in different agro-ecological zones under different production system.

Genetic characterization of poxviruses in Camelus dromedarius in Ethiopia, 2011-2014.

Camelpox and camel contagious ecthyma are infectious viral diseases of camelids caused by camelpox virus (CMLV) and camel contagious ecthyma virus (CCEV), respectively. Even though, in Ethiopia, pox disease has been creating significant economic losses in camel production, little is known on the responsible pathogens and their genetic diversity. Thus, the present study aimed at isolation, identification and genetic characterization of the causative viruses. Accordingly, clinical case observations, infectious virus isolation, and molecular and phylogenetic analysis of poxviruses infecting camels in three regions and six districts in the country, Afar (Chifra), Oromia (Arero, Miyu and Yabello) and Somali (Gursum and Jijiga) between 2011 and 2014 were undertaken. The full hemagglutinin (HA) and partial A-type inclusion protein (ATIP) genes of CMLV and full major envelope protein (B2L) gene of CCEV of Ethiopian isolates were sequenced, analyzed and compared among each other and to foreign isolates. The viral isolation confirmed the presence of infectious poxviruses. The preliminary screening by PCR showed 27 CMLVs and 20 CCEVs. The sequence analyses showed that the HA and ATIP gene sequences are highly conserved within the local isolates of CMLVs, and formed a single cluster together with isolates from Somalia and Syria. Unlike CMLVs, the B2L gene analysis of Ethiopian CCEV showed few genetic variations. The phylogenetic analysis revealed three clusters of CCEV in Ethiopia with the isolates clustering according to their geographical origins. To our knowledge, this is the first report indicating the existence of CCEV in Ethiopia where camel contagious ecthyma was misdiagnosed as camelpox. Additionally, this study has also disclosed the existence of co-infections with CMLV and CCEV. A comprehensive characterization of poxviruses affecting camels in Ethiopia and the full genome sequencing of representative isolates are recommended to better understand the dynamics of pox diseases of camels and to assist in the implementation of more efficient control measures.

Molecular characterization of orf virus from sheep and goats in Ethiopia, 2008-2013.

BACKGROUND: Orf is a contagious disease of sheep, goats and wild ungulates caused by orf virus (ORFV) a member of the genus Parapoxvirus, Poxviridae family. Although orf is endemic in Ethiopia, little attention has been given so far as it is not a notifiable disease by the World Organization for Animal Health. In this work, we have investigated orf outbreaks representing five different geographical locations of Ethiopia, in Amba Giorgis, Gondar zuria, Adet, Debre zeit and Adami Tulu, between 2008 and 2013. RESULTS: The viral isolation and the sequence analysis of the A32L and the B2L genes of eighteen representative isolates confirmed that sampled animals were infected by ORFVs. The phylogenetic study and the comparative analysis of the deduced amino acid profile suggests that there were two main clusters of ORFV isolates which were responsible for the investigated outbreaks. Additionally the analysis of these two genes showed limited variability to ORFVs encountered elsewhere. This is the first report on the genetic characterization of the ORFV isolates from sheep and goats in Ethiopia. CONCLUSION: The molecular characterization of Ethiopian ORFV isolates highlighted the circulation of two main clusters causing orf disease in sheep and goats. The use of laboratory based methods and a constant monitoring of Ethiopian ORFV isolates is needed to better understand the dynamic of ORFV circulating in the country and facilitate the implementation of control measures.

Capripox disease in Ethiopia: Genetic differences between field isolates and vaccine strain, and implications for vaccination failure.

Sheeppox virus (SPPV), goatpox virus (GTPV) and lumpy skin disease virus (LSDV) of the genus Capripoxvirus (CaPV) cause capripox disease in sheep, goats and cattle, respectively. These viruses are not strictly host-specific and their geographical distribution is complex. In Ethiopia, where sheep, goats and cattle are all affected, a live attenuated vaccine strain (KS1-O180) is used for immunization of both small ruminants and cattle. Although occurrences of the disease in vaccinated cattle are frequently reported, information on the circulating isolates and their relation to the vaccine strain in use are still missing. The present study addressed the parameters associated with vaccination failure in Ethiopia. Retrospective outbreak data were compiled and isolates collected from thirteen outbreaks in small ruminants and cattle at various geographical locations and years were analyzed and compared to the vaccine strain. Isolates of GTPV and LSDV genotypes were responsible for the capripox outbreaks in small ruminants and cattle, respectively, while SPPV was absent. Pathogenic isolates collected from vaccinated cattle were identical to those from the non-vaccinated ones. The vaccine strain, genetically distinct from the outbreak isolates, was not responsible for these outbreaks. This study shows capripox to be highly significant in Ethiopia due to low performance of the local vaccine and insufficient vaccination coverage. The development of new, more efficient vaccine strains, a GTPV strain for small ruminants and a LSDV for cattle, is needed to promote the acceptance by farmers, thus contribute to better control of CaPVs in Ethiopia.

Sequence variability in the structural protein-encoding region of foot-and-mouth disease virus serotype A and O of Ethiopian isolates.

A total of 13 serotype O and 5 serotype A FMD Ethiopian isolates and some isolates from other countries (six for serotype A and four for serotype O) were sequenced on the structural protein (P1) coding region. The deduced amino acid sequences were aligned and investigated in an attempt to determine the amino acid variation. Differences were observed at 115 (15.6%) and 119 (16.1%) amino acid positions for serotype O and serotype A, respectively. The variation in the derived amino acid sequences is the highest in VP1, while VP4 was highly conserved in both serotypes A and O. In all isolates, hypervariable regions were located at regions corresponding to the highly immunogenic sites, the G-H loop (133-158) and the C-terminus (194-213) of the VP1 gene. The RGD cell attachment site within the G-H loop of the gene was conserved in all isolates. The study revealed the presence of significant amino acid variation at VP2 and VP3 in addition to known VP1 coding region. Hence, determination of amino acid sequence of the whole P1 region provides more information on antigenic variability of FMD virus and could be used in vaccine strain selection in parallel with serological vaccine matching assays.

An international collaborative study to determine the prevalence of contagious caprine pleuropneumonia by monoclonal antibody-based cELISA.

BACKGROUND: Few serological tests are available for detecting antibodies against Mycoplasma capricolum subsp. capripneumoniae, the causal agent of contagious caprine pleuropneumonia (CCPP). The complement fixation test, the test prescribed for international trade purposes, uses a crude antigen that cross-reacts with all the other mycoplasma species of the "mycoides cluster" frequently infecting goat herds. The lack of a more specific test has been a real obstacle to the evaluation of the prevalence and economic impact of CCPP worldwide. A new competitive ELISA kit for CCPP, based on a previous blocking ELISA, was formatted at CIRAD and used to evaluate the prevalence of CCPP in some regions of Kenya, Ethiopia, Mauritius, Tajikistan and Pakistan in an international collaborative study. RESULTS: The strict specificity of the test was confirmed in CCPP-free goat herds exposed to other mycoplasma species of the "mycoides cluster". Prevalence studies were performed across the enzootic range of the disease in Africa and Asia. Seroprevalence was estimated at 14.6% in the Afar region of Ethiopia, whereas all the herds presented for CCPP vaccination in Kenya tested positive (individual seroprevalence varied from 6 to 90% within each herd). In Mauritius, where CCPP emerged in 2009, nine of 62 herds tested positive. In Central Asia, where the disease was confirmed only recently, no positive animals were detected in the Wakhan District of Afghanistan or across the border in neighboring areas of Tajikistan, whereas seroprevalence varied between 2.7% and 44.2% in the other districts investigated and in northern Pakistan. The test was also used to monitor seroconversion in vaccinated animals. CONCLUSIONS: This newly formatted CCPP cELISA kit has retained the high specificity of the original kit. It can therefore be used to evaluate the prevalence of CCPP in countries or regions without vaccination programs. It could also be used to monitor the efficacy of vaccination campaigns as high-quality vaccines induce high rates of seroconversion.

Genetic characterisation of infectious bursal disease virus isolates in Ethiopia.

The objective of the investigation was to characterise infectious bursal disease viruses (IBDV) circulating in commercial and breeding poultry farms in Ethiopia between 2009 and 2011. The nucleotide and deduced amino acid sequence for VP2 hypervariable region of ten IBDVs were determined by RT-PCR, sequenced and compared to well characterised IBDV isolates worldwide. IBDV genetic material was amplified directly from bursa or cell passaged material. Phylogenetically, Ethiopian IBDVs represented two genetic lineages: very virulent (vv) IBDVs or variants of the classical attenuated vaccine strain (D78). The nucleotide identity between Ethiopian vvIBDVs ranged between 0% and 2.6%. Ethiopian vvIBDVs are clustered phylogenetically with the African IBDV genetic lineage, independent of the Asian/European lineage. This report demonstrates the circulation of vvIBDV in commercial and breeding poultry farms in Ethiopia.

The first isolation and molecular characterization of camelpox virus in Ethiopia.

A cross-sectional study was conducted from November 2011 to April 2012 in Chifra district of Afar and in Jigjiga Zone of Somali Regional States of Ethiopia with the aims of assessing the epidemiology of camelpox and isolate and molecularly characterize the virus. The study included a questionnaire, active disease search and virus isolation and sequencing. A total of 24 (4.50%) and 12 (3.0%) camels in Afar and Jigjiga respectively were found clinically sick of camelpox during the study period. The questionnaire survey indicated that camelpox is the most common disease in the areas in which 125 (96%) of the respondents reported the frequent occurrence of camelpox in their herds especially during rainy season. The PCR result revealed 12 out of 17 tested samples were positive, of which seven of them collected from Jigjiga zone showed the characteristic PCR positive bands of 881 bp size fragments while five of the Afar samples gave two faint bands. Ethiopian isolates, specially isolated from Somali have very high identity with comparable sequences of CMLV M-96 from Kazakhstan and CMLV CMS from Iran. Out of the total of 780 bp analogous sequences, Ethiopian isolates differ only in two positions, while CMLV-Teheran differed at four nucleotide positions. The successfull isolation and molecular characterization of camelpox virus in Ethiopia, which could help for early diagnosis and control of the disease in the country.

Outbreak investigation and molecular characterization of African horse sickness virus circulating in selected areas of Ethiopia.

The study was conducted from June 2011 to May 2012 in central, northern and western parts of Ethiopia to investigate and identify circulating serotypes of African horse sickness virus (AHSV). The indigenous knowledge of equine owners about AHS in the study areas was assessed and also the retrospective data of AHS outbreaks for 2011 were analyzed. Whole blood samples were collected for virus isolation and serotyping from diseased horses and mules showing typical signs of the AHS. Virus isolation on Vero cell and detection of AHSV genomes using conventional RT-PCR were conducted. Further molecular characterization and serotyping were done on positive isolates. The questionnaire survey revealed that equine owners do recognize AHS clinically and have a local name that varies in different regions. From the 72 equine owners interviewed about their knowhow of AHS, 48 (66.7%) of respondents were not aware of AHS disease mode of transmission. The retrospective disease report data showed that a total of 208 outbreaks were reported and 3036 cases and 1167 deaths were recorded in 2011. AHS outbreaks were more frequently observed from September to December and the highest number of outbreaks was recorded in October. During the study period totally six outbreaks were investigated and a total of 62 horses and 10 mules were found sick and all the four forms of AHS were observed. Cardiac form accounted for 52.8%, followed by African horse sickness fever form 31.9%, pulmonary form 8.4% and mixed form 6.9%. AHSV-9 was the only serotype circulating in the outbreak areas.

First confirmation of foot and mouth disease virus serotype SAT-1 in cattle and small ruminants in Ethiopia in 2007/08.

The study was conducted in three regional states of Ethiopia: Amhara, Oromia, and the Southern Nations Nationalities and people regional state from August 2007 to April 2008 with the objective of identifying the foot and mouth disease virus (FMDV) serotypes circulating in the region. Two serotypes were recorded from epithelial tissue and oesophageal-pharyngeal (OP) fluid that were taken from outbreaks in study regions of Ethiopia. Serotype O FMDV was identified in Girar Jarso, Yabello, and Ankesha Guagusa districts while SAT-1 was isolated in Surma and Maji districts from tissue samples and this was the first report of the FMDV serotype in Ethiopia. Similarly, the OP fluid samples were found positive for SAT-1 FMDV in Maji and Surma districts.

Gross and histopathological studies on pulmonary lesions of camel (Camelus dromedarius) slaughtered at Addis Ababa abattoir, Ethiopia.

This study was carried out with the aim of identifying types of gross and histopathological lesions in lungs of camels slaughtered between October 2009 and April 2010 at Addis Ababa abattoir enterprise, Ethiopia. All camels were originated from Borana and Kereyu areas. A total of 387 slaughtered camel lungs were inspected during the study period. Of which, one or more gross lesions were encountered on 300 lungs. Lesions were further subjected for detail gross and histopathological examinations. The occurrence of pulmonary lesions was 77.5%. The gross and histopathological examination of these lesions had revealed 60.2% emphysema, 21.2% hydatidosis, 18.6% pneumonia, 10.6% atelectasis, 4.9% aspiration of blood, 3.9% pneumoconiosis, 2.6% pulmonary edema and congestion, 1.6% abscess, 1% pleurisy, and 0.8% granulomatous pneumonia. Most camels had one or more pulmonary lesions on postmortem examination, but they were apparently healthy during antemortem inspection. Therefore, the prevailing stressful environmental condition coupled with the existing poor level of veterinary service in camel-rearing areas of the country might reverse these hidden inactive lesions and thereby contributed for the higher occurrence of respiratory diseases in camels.

Serological survey of African horse sickness in selected districts of Jimma zone, Southwestern Ethiopia.

A cross-sectional serological survey was undertaken in selected districts of different agro-ecology of Jimma zone (Dedo, Yebu, Seka, Serbo, and Jimma town) from November 2009 to February 2010 to determine the seroprevalence of African horse sickness virus and associated risk factors of the disease. Two hundred seventy-four equids (189 horses, 43 mules, and 47 donkeys) with a history of non-vaccination for at least 2 years were selected randomly from the above areas. Sera samples were collected and assayed for the presence of specific antibody against African horse sickness virus using blocking ELISA. An overall seroprevalence of 89 (32.5%) was found and it was 24 (51.1%) for donkeys, 13 (30.2%) for mules, and 52(28.3%) for horses. Seroprevalence was significantly (X(2) = 11.05, P < 0.05) different among the different species of equids. Seroprevalence was also significantly (X(2) = 11.43, P < 0.05) different among the different agro-ecological areas being higher in highlands 47 (40.5%) followed by midland 30 (34.5%) and lowland 12 (16.9%). Age and sex were not significantly (X(2) = 3.15, P > 0.05 and X(2) = 3.38, P > 0.05, respectively) associated with seroprevalence of AHSV. The present study showed that African horse sickness (AHS) is highly prevalent disease for the horses followed by mules and then donkeys in Jimma zone explained by lower seroconversion rate. Therefore, control strategy against AHS should target at high risk species of all age and sex in their locality in the initial stage for better containment of the disease.

Outbreak investigations and genetic characterization of foot-and-mouth disease virus in Ethiopia in 2008/2009.

The study was conducted in three regional states of Ethiopia: Amhara, Oromia, and Addis Ababa from August 2008 to April 2009 with the objectives of identifying the genetic diversity of serotypes and topotypes in Ethiopia, and determining the attack rate and associations of potential risk factors with foot-and-mouth disease (FMD) seropositivity. A total of 496 cattle were clinically and serologically examined for presence of specific lesions and nonstructural protein for FMD, respectively. Of which, 140 (28.2%) manifested clinical signs and lesions suggestive of FMD, and 219 (44.2%) were seropositive. From a total of 7,781 animals observed and recorded on a designed format in six districts, 1,409 (19.6%) were infected, and 15 (0.12%) died during outbreaks of FMD. Epidemiological investigations revealed that the morbidity rate of the disease was 21.1% in Akaki-kality sub-city, but the mortality rate was <2% in all districts. Furthermore, the mortality and case fatality rates were relatively higher, 1.6% and 8.9% in calves than the other age groups, respectively. From a total of 33 bovine epithelial tissue-cultured samples, 19 (57.6%) showed CPE for FMD virus, in which 16 samples had serotype O and EA-3 topotype, while three samples had found serotype A, Africa topotype, and G-VII strain. Various strains of FMD viruses were isolated in Ethiopia in this study, and therefore, further detailed studies on the evaluation of available vaccines and the development of a vaccine which contains cocktails of antigens of FMD virus strains in the country should be encouraged.