Characterization of Avian Influenza and Newcastle Disease Viruses from Poultry in Libya.

On March 2013, the Libyan poultry industry faced severe outbreaks due to mixed infections of APMV-1 (Newcastle disease) and low pathogenic avian influenza (AI) of the H9N2 subtype which were causing high mortality and great economic losses. APMV-1 and H9N2 were isolated and characterized. Genetic sequencing of the APMV-1/chicken/Libya/13VIR/ 7225-1/2013 isolate revealed the presence of a velogenic APMV-1 belonging to lineage 5 (GRRRQKR*F Lin.5) or genotype VII in class II, according to the nomenclature in use. Three AI viruses of the H9N2 subtype, namely A/avian/Libya/13VIR7225-2/2013, A/avian/Libya/13VIR7225-3/2013, and A/avian/Libya/13VIR7225-5/2013, were isolated and found to belong to the G1 lineage. Analysis of amino acid sequences showed that the analyzed H9N2 viruses contained the amino acid Leu at position 226 (H3 numbering) at the receptor binding site of the HA, responsible for human virus-like receptor specificity. On March 2014, an outbreak of highly pathogenic avian influenza (HPAI) virus of the H5N1 subtype was diagnosed in a backyard poultry farm in an eastern region of Libya. The H5N1 isolate (A/chicken/Libya/14VIR2749-16/2014) was detected by real time RT-PCR (rRT-PCR). Genetic characterization of the HA gene revealed that the identified subtype was highly pathogenic, belonged to the 2.2.1 lineage, and clustered with recent Egyptian viruses. This study revealed the presence of a velogenic APMV-1 genotype and of two influenza subtypes, namely HPAI H5N1 and H9N2, which are of major interest for public and animal health. Considering these findings, more investigations must be undertaken to establish and implement adequate influenza surveillance programs; this would allow better study of the epidemiology of APMV-1 genotype VII in Libya and evaluation of the current vaccination strategies.

The first evidence of bovine viral diarrhea virus circulation in Libya.

Background and Aim: Bovine viral diarrhea (BVD) is endemic in North Africa and the Mediterranean Basin with high socioeconomic impacts. However, there are no data on this disease in Libya. One of the aims of this study was to provide data on BVD in Libya, to fill in the gap in the region and to investigate the level of seroprevalence of BVD virus (BVDV) in Libya and associated risk factors. Material and Methods: A total of 1599 serum samples were collected from cattle herds belonging to seven Libyan regions. All sera were assayed using a screening enzyme-linked immunosorbent assay for the detection of antibodies against BVDV. Results: The overall seroprevalence of BVDV was estimated to be 48.6% (95% confidence interval, 46.08%-50.98%). A seroprevalence rate of 36.8% was detected in cattle aged <1 year, 41.0% in cattle aged between 1 and 2 years, and 49.7% in cattle aged >2 years. Statistically significant differences (p = 0.001) were observed between age groups. BVDV seroprevalence was significantly associated with geographical region (p = 0.033). Conclusion: To the best of our knowledge, this is the first study on BVD in Libya, and the results suggest that BVD is endemic in Libya. Further studies are required to isolate and characterize the circulated BVDV in Libya.

Efficacy of Fowlpox Virus Vector Vaccine Expressing VP2 and Chicken Interleukin-18 in the Protection against Infectious Bursal Disease Virus.

In mammals, the role of interleukin-18 (IL-18) in the immune response is to drive inflammatory and, normally therefore, anti-viral responses. IL-18 also shows promise as a vaccine adjuvant in mammals. Chicken IL-18 (chIL-18) has been cloned. The aim of this study was to investigate the potential of chIL-18 to act as a vaccine adjuvant in the context of a live recombinant Fowlpox virus vaccine (fpIBD1) against Infectious bursal disease virus (IBDV). fpIBD1 protects against mortality, but not against damage to the bursa of Fabricius caused by IBDV infection. The Fowlpox virus genome itself contains several candidate immunomodulatory genes, including potential IL-18 binding proteins (IL-18bp). We knocked out (Δ) the potential IL-18bp genes in fpIBD1 and inserted (::) the cDNA encoding chIL-18 into fpIBD1 in the non-essential ORF030, generating five new viral constructs -fpIBD1::chIL-18, fpIBD1ΔORF073, fpIBD1ΔORF073::chIL-18, fpIBD1ΔORF214, and fpIBD1ΔORF214::chIL-18. The subsequent protection from challenge with virulent IBDV, as measured by viral load and bursal damage, given by these altered fpIBD1 strains, was compared to that given by the original fpIBD1. Complete protection was provided following challenge with IBDV in chicken groups vaccinated with either fpIBDIΔ073::IL-18 or fpIBD1Δ214::IL-18, as no bursal damage nor IBDV was detected in the bursae of the birds. The results show that chIL-18 can act as an effective vaccine adjuvant by improving the fpIBD1 vaccine and providing complete protection against IBDV challenge.

Evidence of West Nile Virus Circulation in Horses and Dogs in Libya.

West Nile virus (WNV) is a globally significant mosquito-borne Flavivirus that causes West Nile disease (WND). In Libya, evidence of WNV circulation has been reported in humans but never in animals. The aim of this study was to determine the seroprevalence of WNV infection in horses and dogs in Libya. In total, 574 and 63 serum samples were collected from apparently healthy, unvaccinated horses and dogs, respectively, between 2016 and 2019. A commercially available competitive enzyme-linked immunosorbent assay (c-ELISA) kit was initially used to test the collected samples for the presence of WNV Ig-G antibodies. Positive and doubtful sera were also tested using a more specific virus neutralisation assay to confirm whether the ELISA-positive results were due to WNV or other Flavivirus antibodies. The seroprevalence of WNV IgG antibodies according to ELISA was 13.2% out of 574 of total horses' samples and 30.2% out of 63 of total dogs' samples. The virus neutralisation test (VNT) confirmed that 10.8% (62/574) and 27% (17/63) were positive for WNV-neutralising titres ranging from 1:10 to 1:640. Univariable analysis using chi-square tests was conducted to measure the statistical significance of the association between the hypothesized risk factors including city, sex, breed, and age group and were then analyzed using the subsequent multivariable logistic regression model for horse samples. Age group was found to be the only significant risk factor in this study. The results of the present study provide new evidence about WNV circulation in Libya.

Results of the eighteenth winter waterbird census in Libya in 2022.

Background: Libyan wetlands are diverse; the coastline of Libya, in particular, has different kinds of wetlands, such as salt marshes, bays, lakes, lagoons, and islands. These varieties in habitats provide good shelters and foraging sites for migratory birds during their journeys between Eurasia and Africa. Since the beginning of the Libyan winter census of waterbirds International waterbirds census (Libya IWC) in 2005, which continued regularly until 2012, it has had relatively the same performance in the number of covered sites. However, since 2013, due to the security situation that Libya has experienced due to wars and conflict, which negatively affected the quality of the IWC in Libya, the number of sites has dramatically decreased, reaching only six locations during the middle of the previous decade. Aim: The IWC 2022 aimed to count the birds along the Libyan coast from January 10 to 29. Methods: The census activities were conducted from dawn to dusk during the study period by using high-quality Telescopes, binoculars, and digital cameras for the documentation. Point transects method was used to cover the sites. Results: The results of this year showed that a total of 64 sites were covered, and 68 species of waterbirds were counted, with an abundance of 61,850 individuals. During the census period, a total of 52 non-waterbird species found in Wetlands were recorded, and the number of individuals was 14,836 birds. A total of 18 threatened species were observed during this survey, 12 of them are mentioned in the International Union for Conservation of Nature Red List, and nine species are mentioned in the regional activities center of specially protected areas annex II as threatened in the Mediterranean, where the species; Larus audouinii (Payraudeau, 1826), Larus genei (Breme, 1839), and Puffinus yelkouan (Acerbi, 1827) are mentioned in both of them. Conclusion: The lack of the number of ornithologists and bird watchers is still one of the factors affecting the quality of the IWC in Libya, as well as lack of funding remains an important factor that plays a major role in the success of the waterbirds census.

FTA Cards as a Rapid Tool for Collection and Transport of Infective Samples: Experience with Foot-and-Mouth Disease Virus in Libya.

Foot-and-mouth disease (FMD) is a viral disease, widespread and highly contagious, that mainly affects cloven-hoofed domestic and wild animals. FMD can lead to high economic losses due to the reduction in animal production such as a drop in milk production, loss of body weight, and a high mortality rate in young ruminants. Sixteen samples were collected from animals showing typical clinical signs of FMD during the last FMD outbreak in Libya in 2018-2019. Flinders Technology Associates (FTA) cards impressed with blood, swabs, or vesicular epithelium samples were shipped to the WOAH FMD reference laboratory in Brescia, Italy, and tested for the detection of FMD viruses. Nucleic acids were extracted from the FTA cards, and molecular testing based on real-time RT-PCR assays was carried out, of which one was specifically designed for the detection of the FMD virus of serotype O, topotype O/East Africa-3 (O/EA-3), that was further confirmed by a sequence analysis of the VP1 gene. The phylogenetic analysis of the VP1 gene showed a nucleotide identity of more than 99% between the virus circulating in Libya and the FMD virus strains isolated in Algeria in 2019.

Identification of diffusion routes of O/EA-3 topotype of foot-and-mouth disease virus in Africa and Western Asia between 1974 and 2019 - a phylogeographic analysis.

Foot-and-mouth disease (FMD) affects the livestock industry and socioeconomic sustainability of many African countries. The success of FMD control programs in Africa depends largely on understanding the dynamics of FMD virus (FMDV) spread. In light of the recent outbreaks of FMD that affected the North-Western African countries in 2018 and 2019, we investigated the evolutionary phylodynamics of the causative serotype O viral strains all belonging to the East-Africa 3 topotype (O/EA-3). We analyzed a total of 489 sequences encoding the FMDV VP1 genome region generated from samples collected from 25 African and Western Asian countries between 1974 and 2019. Using Bayesian evolutionary models on genomic and epidemiological data, we inferred the routes of introduction and migration of the FMDV O/EA-3 topotype at the inter-regional scale. We inferred a mean substitution rate of 6.64 × 10-3  nt/site/year and we predicted that the most recent common ancestor for our panel of samples circulated between February 1967 and November 1973 in Yemen, likely reflecting the epidemiological situation in under sampled cattle-exporting East African countries. Our study also reinforces the role previously described of Sudan and South Sudan as a frequent source of FMDVs spread. In particular, we identified two transboundary routes of O/EA-3 diffusion: the first from Sudan to North-East Africa, and from the latter into Israel and Palestine AT; a second from Sudan to Nigeria, Cameroon, and from there to further into West and North-West Africa. This study highlights the necessity to reinforce surveillance at an inter-regional scale in Africa and Western Asia, in particular along the identified migration routes for the implementation of efficient control measures in the fight against FMD.

Exploiting epidemiological data to understand the epidemiology and factors that influence COVID-19 pandemic in Libya.

There were only 75 confirmed cases of coronavirus disease 2019 (COVID-19) reported in Libya by the National Center for Disease Control during the first two months following the first confirmed case on 24 March 2020. However, there was dramatic increase in positive cases from June to now; as of 19 November 2020, approximately 357940 samples have been tested by reverse transcription polymerase chain reaction, and the results have revealed a total number of 76808 confirmed cases, 47587 recovered cases and 1068 deaths. The case fatality ratio was estimated to be 1.40%, and the mortality rate was estimated to be 15.90 in 100000 people. The epidemiological situation markedly changed from mid-July to the beginning of August, and the country proceeded to the cluster phase. COVID-19 has spread in almost all Libyan cities, and this reflects the high transmission rate of the virus at the regional level with the highest positivity rates, at an average of 14.54%. Apparently, there is an underestimation of the actual number of COVID-19 cases due to the low testing capacity. Consequently, the Libyan health authority needs to initiate a large-scale case-screening process and enforce testing capacities and contact testing within the time frame, which is not an easy task. Advisably, the Libyan health authority should improve the public health capacities and conduct strict hygienic measures among the societies and vaccinate as many people against COVID-19 to minimize both the case fatality ratio and socio-economic impacts of the pandemic in Libya.

Rabies in bovine: First case report of rabies in Al Jabal Al Akhdar, Libya.

Background: Rabies is still one of the most neglected diseases in developing countries. It is endemic to North Africa, although rabies incidence in North Africa is certainly underestimated. Case Description: On 18 December 2018 in the region of Al-Jabal Al-Akhdar, an 8-month-old calf died after a period of characteristic clinical symptoms of rabies.This is the first case of rabies in bovine which is confirmed through histopathological examination in the Laboratory of Veterinary Pathology, Omar Al-Mukhtar University. Microscopic examination clearly revealed encephalitis with the pathognomonic Negri bodies in the cerebellar neurons. Conclusion: Since the characteristic lesions in the histopathological examination are sufficient to confirm the diagnosis and report infected cases, we recommend that the next version of the OIE Terrestrial Manual should add and clarify that the results of the use of histopathological techniques in the diagnosis of rabies are significant.

First seroprevalence investigation of epizootic haemorrhagic disease virus in Libya.

Background: Epizootic haemorrhagic disease (EHD) is a vector-borne viral disease of domestic and wild ruminants. Epizootic haemorrhagic disease virus (EHDV) is transmitted by Culicoides spp. EHDV is a member of the Orbivirus genus within the Reoviridae family. It shares many morphological and structural characteristics with other members of the genus, such as the bluetongue virus, African horse sickness virus, and equine encephalosis virus. Aims: The purpose of our study was to investigate the epidemiological situation of EHDV in Libya in order to gain some knowledge about the presence of this virus in the country. Methods: In this study, we investigated the seroprevalence of EHDV in Libya, testing 855 blood samples collected during 2015. The samples were collected from domestic ruminants (cattle, sheep, and goats) originating from 11 provinces of Libya. Sera were tested by competitive enzyme-linked immunosorbent assays and positive samples confirmed by serum neutralization test. Results: The overall seroprevalence of EHDV was estimated to be 4% (95% confidence intervals = 2.8%-5.4%). Small ruminant seroprevalence was significantly (p = 0.016) higher than that found in cattle. Neutralizing antibodies against EHDV-6 were detected in a sheep from the western region of Libya. Conclusion: This study suggests that EHDV has circulated or is circulating in Libya, and sheep could play an important role in the epidemiology of EHDV, and the virus may still be circulating in North Africa.

Seroprevalence and molecular detection of Newcastle disease virus in backyard chickens in Tripoli, Libya.

Background: Newcastle disease (ND) is a viral disease that affecting many avian species all over the world. Aim: ND has been successfully controlled by the vaccination of commercial poultry in Libya. However, there was a lack of information about the situation of ND in backyard chickens. Therefore, this study determined the prevalence of ND in backyard chickens in different locations of Tripoli. Methods: A total number of 280 cloacal swabs (190 in summer and 90 in winter) and 412 sera were collected from non-vaccinated backyard chicken flocks in different geographical locations within the area of Tripoli namely Qasr Ben Ghashier, Al-Sawani, Souq Al-Gomaa, Tajourah, Ein Zara, and Janzour. Cloacal swabs and sera were tested by real time polymerase chain reaction (PCR) and ELISA, respectively. Results: The prevalence of ND virus (NDV) infection in backyard chickens in different locations of Tripoli during summer and winter was 45% using real-time reverse transcription-PCR. Except in Qasr Ben Ghashier, the prevalence in summer season was significantly higher than in winter (X2 = 46.13, p ≥ 0.00001). ELISA test revealed 218 positive out of 412 tested samples with total prevalence of 53% across the city of Tripoli in all regions. Obviously, Qasr Ben Ghashier had significantly (X2 = 74.09, p ≥ 0.00001) the highest prevalence (82%) of NDV specific antibodies followed by Tajourah (68%). Conclusion: This study demonstrated the situation of ND in backyard chicken highlighting the necessity of a comprehensive vaccination plan for backyard chickens.

Exploiting serological data to understand the epidemiology of bluetongue virus serotypes circulating in Libya.

The epidemiological patterns of Bluetongue (BT) in North Africa and Mediterranean Basin (MB) dramatically changed by emergence of subsequent episodes of novel bluetongue virus (BTV) serotypes with highly pathogenic indexes and socio-economic impacts. The objective of the study was to investigate the sero-prevalence and serotype distribution of BTV in Libya. During 2015-2016, a total of 826 serum samples were collected from domestic ruminants in Libya. All sera were assayed by competitive enzyme-linked immunosorbent assays (c-ELISA). C-Elisa-positive samples (43.3%; 173/400) were further analyzed by virus neutralization assay to identify BTV serotypes and determine the antibody titre of positive samples. An overall BTV sero-prevalence was 48.4% (95% CI: 45.0%-51.8%). Neutralizing antibodies were detected against the following BTV serotypes namely: BTV-1, BTV-2, BTV-3, BTV-4, BTV-9 and BTV-26. While BTV-1, BTV-2, BTV-4 and BTV-9 circulation was unsurprising as they have been responsible of the last year outbreaks in Northern African Countries, the detection of BTV-3 and BTV-26 was definitely new and concerning for the animal health of the countries facing the Mediterranean Basin. It is crucial that European and Northern African authorities collaborate in organizing common surveillance programmes to early detect novel strains or emerging serotypes in order to set up proper preventive measures, and, in case, develop specific vaccines and plan coordinated vaccination campaigns.

Rift Valley fever virus: a serological survey in Libyan ruminants.

A serological survey was carried out in Libya to investigate the circulation of Rift Valley fever virus (RVFV) among domestic ruminants. A total of 857 serum samples were collected from year 2015 to 2016 in eleven provinces of Libya belonging to five branches of the country. Samples were tested for RVFV antibodies using a competitive Enzyme-Linked Immunosorbent Assay (c-ELISA). Antibodies specific for RVFV were not detected in any of the 857 samples. However, a statistical analysis was carried out to assess the maximum expected number of infected animals and the maximum expected prevalence of RVFV among Libyan ruminants' populations according to the sampled population. The overall maximum expected prevalence was estimated to be 1.8% for cattle and 0.4% for small ruminants. Results seem to exclude the circulation of RVFV, however, a surveillance plan should be implemented in areas at risk of RVFV introduction.

Reconstructing the evolutionary history of pandemic foot-and-mouth disease viruses: the impact of recombination within the emerging O/ME-SA/Ind-2001 lineage.

Foot-and-mouth disease (FMD) is a highly contagious disease of livestock affecting animal production and trade throughout Asia and Africa. Understanding FMD virus (FMDV) global movements and evolution can help to reconstruct the disease spread between endemic regions and predict the risks of incursion into FMD-free countries. Global expansion of a single FMDV lineage is rare but can result in severe economic consequences. Using extensive sequence data we have reconstructed the global space-time transmission history of the O/ME-SA/Ind-2001 lineage (which normally circulates in the Indian sub-continent) providing evidence of at least 15 independent escapes during 2013-2017 that have led to outbreaks in North Africa, the Middle East, Southeast Asia, the Far East and the FMD-free islands of Mauritius. We demonstrated that sequence heterogeneity of this emerging FMDV lineage is accommodated within two co-evolving divergent sublineages and that recombination by exchange of capsid-coding sequences can impact upon the reconstructed evolutionary histories. Thus, we recommend that only sequences encoding the outer capsid proteins should be used for broad-scale phylogeographical reconstruction. These data emphasise the importance of the Indian subcontinent as a source of FMDV that can spread across large distances and illustrates the impact of FMDV genome recombination on FMDV molecular epidemiology.

An epidemological study on Peste des petits ruminants in Tripoli Region, Lybia.

A cross­sectional study was conducted in Libya in 7 areas of Tripoli to determine the seroprevalence of Peste des Petits Ruminants (PPR) Virus (PPRV) in small ruminants (sheep and goats) between June and August 2013, and to identify the potential risk factors associated with the infection. The study involved 10% of small ruminant herds with ≥50 animals in the Tripoli region. They were selected randomly (15 herds), and 35 to 58 samples, depending on its size, were collected from each selected herd. Seven­hundred and twenty­one serum samples from unvaccinated animals (601 of sheep and 120 of goats) were collected and then tested using cELISA commercial kit in the National Center of Animal Health Laboratory in Tripoli, Libya. The overall seroprevalence was 46.7% [(sheep 44.3% (266/601) and goats 59.2% (71/120)]. Mean within­herd prevalence was 48.5% (95% CI: 32.1% ­ 64.8%), and the herd prevalence was 93.3% (14/15). Various risk factors at the animal and herd levels were analysed by multivariable logistic regression model (forward stepwise). The results identified breed, source of animal, and region as significant risk factors (p< 0.05). As for the source of new animal to the farm, PPRV seroprevalence was highest in illegally imported animals (90.9%), followed by the seroprevalence in animal legitimately acquired (55.8%), and by the seroprevalence in animals belonging to the same herd (4.7%). The seroprevalence among breeds was 69.5% (228÷328) in illegally imported animals, whereas 27.7% (109÷393) was found to be in local breed. Seroprevalence in the areas considered in this study was higher (66.2%) in Al­Mashroa area followed by Ein­zara (57.8%), Arada (50%), Ben­Own (47%), AL­Naem (37.5), Ber­Alalem (24.5) and in Tajora (0%). The results indicated that PPRV virus was actively circulating in Tripoli regions and that the illegal importing of animals was the main source of spreading PPR in Tripoli regions, showing that better efforts should be made to raise public awareness with respect to biosecurity.

Biosecurity and geospatial analysis of mycoplasma infections in poultry farms at Al-Jabal Al-Gharbi region of Libya.

Geospatial database of farm locations and biosecurity measures are essential to control disease outbreaks. A study was conducted to establish geospatial database on poultry farms in Al-Jabal Al-Gharbi region of Libya, to evaluate the biosecurity level of each farm and to determine the seroprevalence of mycoplasma and its relation to biosecurity level. A field team of 7 Veterinarians belongs to the National Center of Animal Health was assigned for data recording and collection of blood samples. Personal information of the producers, geographical locations, biosecurity measures and description of the poultry farms were recorded. The total number of poultry farms in Al-Jabal Al-Gharbi Region is 461 farms distributed in 13 cities. Out of these, 102 broiler farms and one broiler breeder farm (10 houses) which were in operation during team visit were included in this study. Following collection of blood, sera were separated and tested by Enzyme-linked immunosorbent assay (ELISA) for the presence of antibodies against Mycoplasma (General antigen for M. gallisepticum and M. synoviae). The seroprevalence of Mycoplasma in the region was 28% (29 poultry farms out of 103 were infected). About 50% (23 out of 47) of poultry farms located in Garian city were infected with Mycoplasma and one significant cluster of Mycoplasma infection in the city was identified. Low level of biosecurity was found in poultry farms of the region. Out of the 103 farms included, 63% of poultry houses has a ground of soil and 44% of them has uncoated walls which may influence the proper cleaning and disinfection. Almost 100% of the farms are at risk of exposure to diseases transmitted by wild birds such as avian influenza and Newcastle disease due to absence of wild birds control program. Although, 81% of the farms have entry restrictions, only 20% have disinfectants at entry which increase the risk of exposure to pathogens. The results of this study highlight the weakness points of biosecurity measures in poultry farms of Al-Jabal Al-Gharbi region and high seroprevalence of mycoplasma. Data collected in this study will assist the Veterinary authorities to apply effective disease control strategies.

Exploiting serological data to understand the epidemiology of foot-and-mouth disease virus serotypes circulating in Libya.

Sporadic outbreaks of foot-and-mouth disease (FMD) have occurred in Libya for almost fifty years. During the spring of 2013, a countrywide serosurvey was undertaken to assess the level of FMD virus circulation and identify FMD virus serotypes in the country. A total of 4221 sera were collected, comprising samples from large ruminants (LR; n=1428 samples from 357 farms) and small ruminants (SR; n=2793 samples from 141 farms). FMD sero-prevalence of NSP antibodies determined by ELISA were 19.0% (271/1428) with 95% CI (16.9 - 21.0) and 13.5% (378/2793) with 95% CI (12.3 - 14.8) for LR and SR samples, respectively. The sero-prevalence of NSP antibodies in LR was 12.3% and 19.8% for age group < 1 year and ≥ 1 year, respectively (X2= 4.95, P= 0.026), while in SR was 3.7%, 13.6% and 21.3% for age group < 1 year, 1-2 year and > 2 year, respectively (X2= 118.1, P= 0.000). These observed NSP serologic profiles support the hypothesis of an endemic level of FMD circulation in Libya. All positive sera were tested for SP antibodies for O, A and SAT-2 FMD virus serotypes. Serotype O was the dominant circulating serotype followed by serotype A, while evidence of SAT-2 was not found. These data provide an insight into the wider epidemiology of FMD in Libya, and contribute to field and laboratory investigations that during 2013 serotype O (O/ME-SA/Ind-2001 lineage) was isolated from clinical samples collected from the country.