Central Veterinary Laboratory Research Articles

Genome characterization of Rift Valley fever virus isolated from cattle, goats and sheep during interepidemic periods in Kenya

Rift Valley fever virus (RVFV) is a mosquito-borne RNA virus of the Phlebovirus genus in the phenuviridae family. Its genome is trisegmented with small (S), medium (M) and large (L) fragments. In nature, the virus exists as a single serotype that is responsible for outbreaks of Rift Valley fever (RVF), a zoonotic disease that often occurs in Africa and the Middle East. RVFV genomes are thought to undergo both recombination and reassortment and investigations of these events is important for monitoring the emergence of virulent strains and understanding the evolutionary characteristics of this virus. The aim of this study was to characterize the genomes of RVFV isolates from cattle, sheep, and goats collected during an interepidemic period in Kenya between June 2016 and November 2021. A total of 691 serum samples from cattle (n = 144), goats (n = 185) and sheep (n = 362) were analysed at the Central Veterinary Laboratories. The competitive IgM-capture ELISA, was used to screen the samples; 205 samples (29.67%) tested positive for RVFV. Of the 205 positive samples, 42 (20.5%) were from cattle, 57 (27.8%) from goats, and 106 (51.7%) from sheep. All the IgM-positive samples were further analyzed by qPCR, and 24 (11.71%) tested positive with Ct values ranging from 14.788 to 38.286. Two samples, 201808HABDVS from sheep and 201810CML3DVS from cattle, had Ct values of less than 20.0 and yielded whole genome sequences with 96.8 and 96.4 coverage, respectively. There was no statistically significant evidence of recombination in any of the three segments and also phylogenetic analysis showed no evidence of reassortment in the two isolated RVFV segments when compared with other isolates of different lineages from previous outbreaks whose genomes are deposited in the GenBank. No evidence of reassortment leaves room for other factors to be the most probable contributors of change in virulence, pathogenicity and emergence of highly virulent strains of the RVFV.

William Albert (Bill) Watson.

After working in general practice, he joined the Veterinary Investigation Service and went on to become director of the government's central veterinary laboratory headquarters.

Isolation and Characterization of Lactobacillus Strains with Probiotic Potential from Dairy Products

Aims: This research aims to isolate and characterize from dairy products, in particular yogurts, cultivated Lactobacillus strains with potential for use as probiotics in poultry farming.
 Study Design: This research consists of four parts, an introduction with literature review, a description of the materials and methods used, finally the results obtained and their discussion.
 Place and Duration of Study: Central Veterinary Laboratory of Kinshasa, specifically in the Bacteriology and Animal Services departments, between April and September 2022.
 Methodology: A yogurt was cultured in order to observe the presence of Lactobacilli in the MRS (Mans, Rogosa and Sharp) Agar culture medium; this culture revealed the growth of Lactobacilli. It was even proceeded to the serial dilution of these strains, the results had also revealed the growth of Lactobacilli in the 6 test tubes used.
 Results: The results obtained showed a significant increase in weight (positive effect on the immune system) in the poultry of the experimental group and an absence of pathogenicity after an incubation period (inhibitory effect of strains of Lactobacilli on those of Escherichia coli and Streptococcus sp.) whereas in the control group, the poultry showed a lower weight gain compared to the experimental group and developed colibacillosis associated with other effects such as weakness, fever and angina after an incubation period.
 Conclusion: The positive results of Lactobacilli strains obtained in poultry in the experimental group show the potential of these strains to be used as probiotics in poultry farming to improve poultry health through the inhibitory effect of these strains on pathogenic Escherichia coli and Streptococcus sp. This opens up safe alternatives to the use of these strains to fight the misuse of antibiotics by reducing their use in human and animal care.

Study on Management Status and Disease aspects of Pheasant (Phasianus colhicus) of Kathmandu Valley, Nepal

Even though Nepal has several wild pheasant species, captive pheasant rearing for human consumption is a relatively new farming practice in Nepal. Pheasant farming is a growing practice among Nepalese farmers. However, farmers lack understanding and background in pheasant management techniques and associated diseases. The purpose of this study was to investigate the distribution, location, management status, and disease characteristics of commercial pheasant farming in the Kathmandu valley. A retrospective cross-sectional study was conducted in Kathmandu valley and the Central Veterinary Laboratory (CVL), from July to September 2021. The case records from 2017 to 2021 were analyzed at CVL and the farms were mapped based on their locations. The top ten pheasant diseases reported in CVL were identified and the disease outbreak farms were followed to get additional information on management aspects. A total of 42 pheasant farms were visited to collect data, and dead birds from these pheasant farms were submitted to CVL for postmortem and bacterial culture. MS-EXCEL and the online software “Open Epi” were used for statistical analysis. Sixty percent of the surveyed farms exclusively raised pheasants, while 40 percent practiced mixed farming. The source of hatching eggs was from abroad (12%), local breeding center (70%) and inter-district farms (18%). Ninety percent of the farms practiced some extent of biosecurity such as clothes changes and visitors log maintained. The temporal analysis identified that pheasant mortality was highest in July, followed by June and August. Of the total cases reported in retrospective study urolithiasis was responsible for 20.27 percent of deaths, Newcastle disease and Mycotoxicosis each were responsible for 13.51 percent of deaths of birds. Though urolithiasis is one of the leading causes of death there was no significant statistical associations (p>0.05) with ages and the flock size. The study collected the baseline data on management practices and disease distributions among the commercial pheasant farming in Kathmandu valley. We suggest, further studies on morbidity, mortality, seasonal stress, and economic aspects of commercial pheasant farming. Prior knowledge and expertise on management and health aspects related to farming is needed for sustainable pheasant farming in Nepal.

From Field Tests to Molecular Tools-Evaluating Diagnostic Tests to Improve Rabies Surveillance in Namibia.

Rabies is endemic in Namibia and is present both in wildlife carnivores and domestic free-roaming dogs. The disease thus represents a challenge for public human and veterinary disease control. Namibia has implemented a national strategic plan to control rabies and the country's activities are supported by international organizations. To this end, rabies diagnosis at the Central Veterinary Laboratory (CVL) was improved in the frame of a World Organization for Animal Health (WOAH) laboratory twinning program: from practical sampling techniques and the use of lateral flow devices to a novel universal and discriminatory quantitative real-time Reverse transcription polymerase chain reaction (RT-qPCR), which easily identify dog-associated rabies viruses. The procedures applied and the results can be used as a template to improve rabies laboratory diagnosis.

Current status and molecular epidemiology of rabies virus from different hosts and regions in Malawi

Although rabies is endemic in Malawi, there have been no studies in which rabies virus was systematically investigated and characterized in multiple animal hosts in that country. In order to provide molecular epidemiological data on rabies virus in Malawi, 683 suspected rabies case reports from 2008 to 2021 were examined, and 46 (dog = 40, cow = 5, and cat = 1) viable rabies-positive brain samples archived at the Central Veterinary Laboratory (CVL), Lilongwe, Malawi, were analyzed genetically. The results showed an increase in the submission of brain samples from 2008 to 2010, with the highest number of submissions observed in 2020. Of the 683 case reports analyzed for the period under review, 38.1% (260/683) (CI: 34.44 – 41.84) were confirmed by direct fluorescent antibody test. Among the confirmed cases, 65.4% (170/260) (CI: 59.23 – 71.09) were canine rabies. Further, phylogenetic analysis revealed that sequences from different animal hosts clustered together within the Africa 1b lineage, suggesting that the strains circulating in livestock are similar to those in domestic dogs. This finding supports the hypothesis that canine rabies is spilling over to livestock and emphasizes the need for further studies to provide data for effective control of rabies in Malawi.

Prevalence of Campylobacter Species on Cattle Breeding Farms in Zimbabwe.

Infertility remains a challenge in breeding herds in most developing countries. In the current study, 104 penile sheath washes were collected from bulls of different breeds and ages from different cattle breeding farms in Zimbabwe. The samples were submitted to the Central Veterinary Laboratory, Zimbabwe, for screening of Campylobacter species using the polymerase chain reaction (PCR). Based on the PCR results, the animal-level prevalence was 25.96% (range 0-73.98%) and all the positive samples came from four (57.14%) of the 7 herds tested. The current study shows that Campylobacter spp. could be a causative agent in infertility observed in a number of herds in Zimbabwe. Strategies for treatment and control of campylobacteriosis should be enhanced in the country. More research and surveillance are needed to determine the epidemiology of Campylobacter species in Zimbabwean cattle herds.

Capacity Building Efforts for Rabies Diagnosis in Resource-Limited Countries in Sub-Saharan Africa: A Case Report of the Central Veterinary Laboratory in Benin (Parakou).

Rabies has been listed as a priority zoonotic disease in many African countries and the countdown to reach the goal of eliminating dog-mediated human rabies deaths by 2030 means that disease control measures need to be applied fast. In this context, an essential pillar of any national plan to control rabies is the implementation of reliable diagnostic techniques to ensure the success of field surveillance systems. Although many African countries have received international support for the control of rabies–some countries, like Benin, have not received a similar level of support. Indeed, until 2018, Benin was not able to diagnose rabies and rabies diagnosis in animals as well as humans relied solely on observed clinical symptoms. Although the Central Veterinary Laboratory (CVL) of Parakou had the equipment to implement two recommended tests, the lack of specific reagents and skills prevented the implementation of a rabies diagnostic service. Here we present the joint efforts of the national authorities in Benin, intergovernmental agencies, and non-governmental organizations to assess the strengths and weaknesses of the government's rabies control efforts. We have applied the Stepwise Approach toward Rabies Elimination (SARE) analysis, implemented rabies diagnostic capacities at the CVL of Parakou, characterized strains of rabies virus circulating in Benin, and finally integrated an inter-laboratory comparison program.

Retrospective Characterization of Initial Peste des petits ruminants Outbreaks (2008-2012) in the Democratic Republic of the Congo.

Peste des petits ruminants (PPR) is an acute, contagious viral disease of small ruminants, goats and sheep. The Democratic Republic of the Congo (DRC) was a PPR-free country until 2007, although in 2006, scare alerts were received from the east and the southwest of the country, reporting repeated mortalities, specifically in goats. In 2008, PPR outbreaks were seen in several villages in the west, leading to structured veterinary field operations. Blood, swabs and pathological specimens consisting of tissues from lungs, spleens, lymph nodes, kidneys, livers and hearts were ethically collected from clinically infected and/or dead animals, as appropriate, in 35 districts. Epidemiological information relating to major risk factors and socio-economic impact was progressively collected, revealing the deaths of 744,527 goats, which converted to a trade value of USD 35,674,600. Samples from infected and dead animals were routinely analyzed by the Central Veterinary Laboratory at Kinshasa for diagnosis, and after official declaration of PPR outbreaks by the FAO in July 2012, selected tissue samples were sent to The Pirbright Institute, United Kingdom, for genotyping. As a result of surveys undertaken between 2008 and 2012, PPR virus (PPRV)-specific antibodies were detected in 25 locations out of 33 tested (75.7%); PPRV nucleic acid was detected in 25 locations out of 35 (71.4%); and a typical clinical picture of PPR was observed in 23 locations out of 35 (65.7%). Analysis of the partial and full genome sequences of PPR viruses (PPRVs) obtained from lymphoid tissues of dead goats collected in Tshela in the DRC in 2012 confirmed the circulation of lineage IV PPRV, showing the highest homology (99.6−100%) with the viruses circulating in the neighboring countries of Gabon, in the Aboumi outbreak in 2011, and Nigeria (99.3% homology) in 2013, although recent outbreaks in 2016 and 2018 in the western part of the DRC that borders with East Africa demonstrated circulation of lineage II and lineage III PPRV.

Haemoparasites in Cattle at Gbele Reserve, Upper West Region, Ghana

Haemoparasitic diseases is one of the major challenges that hamper cattle production in Africa with their effect usually manifested in production losses, late maturity, weight loss, still birth and increased susceptibility to other diseases. Information on the burden of some haemoparasites in wildlife/livestock interface in Ghana is scanty in literature. This study was carried out to determine the prevalence of haemoparasites in cattle at Gbele Reserve wildlife/livestock interface, Upper west region of Ghana. A total of 143 blood samples collected from four selected fringe communities namely Desima, Gbele, Wahabu and Sakalu were transported to Central Veterinary Laboratory at Pong-Tamale for analysis. Giemsa stained thin blood smears were examined for haemoparasites, and packed cell volume (PCV) for each sample was determined using Haematocrit method. Mean PCV value was 25% and 42% for infected and non-infected cattle respectively. An overall prevalence of haemoparasites was 2.8% for all samples examined. Village specific prevalence of 0.7%, 1.4%, 0.0% and 0.7% was recorded in cattle infected in Gbele, Desima, Sakalu and Wahabu respectively. The low prevalence of haemoparasites recorded was attributed to the earlier intervention by Pan-African Tsetse and Trypanosomosis Eradication campaign (PATTEC) project and the quarterly treatment regime employed by farmers. Babesia bovis and Anaplasma marginale were identified with prevalence of 2.1% and 0.7% respectively. The distribution of parasite based on sex was 2.1% for cows and 0.7% for bulls. Polymerase chain reaction and enzyme-linked immunosorbent assays are recommended alongside with thin blood smear examination in future studies for better sensitivity and identification of the haemoparasites to species level.

Animal rabies epidemiology in Nepal from 2005 to 2017

Background and Aim: Animal rabies is endemic in Nepal, and it occurs in two forms. Although governmental and non-governmental agencies are working toward the control of rabies by mass dog vaccination and stray dog population management, there is still massive number of rabies incidence reported to the reference veterinary laboratory, Nepal. Therefore, this study aimed to assess animal species, temporal, regional, and agro-ecological distribution patterns of animal rabies in Nepal from 2005 to 2017. Materials and Methods: The epidemiological data on animal rabies from the period of 2005 to 2017 were obtained from the Central Veterinary Laboratory, Tripureshwor, Kathmandu, Nepal. The laboratory-confirmed rabies cases were analyzed according to animal species, temporal, regional (developmental zones), and agro-ecological distributions. In addition, descriptive statistics were used to evaluate the distribution patterns of rabies. Results: From 2005 to 2017, a total of 2771 suspected rabies cases in animals were reported to The Central Veterinary Hospital, Kathmandu. Of which, 1302 were found laboratory-confirmed cases. The rabies cases were most commonly reported and confirmed in dogs followed by other domestic animals. The high occurrences were recorded between 2005 and 2007. However, the incidence was increased during 2016 and 2017. The highest number of rabies cases was recorded in the eastern development zone, and the least number in the central zone at regional level. Likewise, it was highest in the Terai (plain) region and lowest in mountainous areas at agro-ecological zones. The findings also revealed that the occurrences of rabies significantly differed among seasons. Conclusion: Rabies is present in Nepal throughout the year and all seasons with seasonal variation. Among the animal species, dogs are the primary animals affected with rabies followed by cattle and other domestic animals. At the regional level, eastern development zone had the highest incidence and Central development zone recorded the least. Similarly, the Terai region had the highest incidence rates, and the least overall prevalence rate was observed in mountainous regions among agro-ecological zones. Therefore, the government should implement the strict enforcement of mass dog vaccination and dog population management through one health approach to control rabies incidence in the country.

Prevalence and Antibiograms of Salmonella in Commercially Produced Crocodile meat in Zimbabwe

The presence of Salmonella in food products and emergence of antibiotic resistance are the major challenges facing public health policies. A total of 2749 crocodile meat samples obtained from the Central Veterinary Laboratories in Zimbabwe were screened for Salmonella specieswere collected from three Zimbabwean commercial farms between the year 2012 and 2019 for a retrospective observational study to determine the prevalence and magnitude of antibiotics resistant Salmonella species in crocodile meat. The isolation of Salmonella was in accordance with the ISO 6579:2002 and the antibiotic susceptibility testing was carried out based on Clinical and Laboratory Standard Institute’s recommendations by means of the Kirby-Bauer disk diffusion method. SILAB Database was used to determine the prevalence of Salmonella species. Prevalence was stratified by year and farms. Twenty Salmonella isolates were identified using biochemical tests, and 15 were confirmed by polymerase chain reaction (PCR). Antimicrobial susceptibility profiles of the confirmed Salmonella isolates were examined using 14 antibiotics. The overall prevalence of Salmonella species in crocodile meat samples was 0.5%. The prevalence of Salmonella species ranged from 0.04% to 0.44% in the crocodile meat samples and annual prevalence ranged from 0.01% to 1%. The highest prevalence of Salmonella (4.4%) was recorded in the year 2012. Salmonella isolates from one of the three tested farms were resistant to Erythromycin (73.33%), Ampicillin (80%), and Penicillin G (100%). Generally, Salmonella isolates displayed lower resistance to Cefepime, Ceftriaxone, Amikacin, Tetracycline, Ertapenem, Florfenicol, and Erythromycin (0-53.33%) whereas all Salmonella isolates showed susceptibility to Cefepime, Ceftriaxone, Ertapenem, and Florfenicol. Although the study indicates low prevalence of Salmonella species in crocodile meat, there is a need for strict implementation of Hazard Analysis Critical Control Point (HACCP) to reduce contamination rates in meat and its products

Investigation of Buffalo Herd’s Diseases-Brucellosis, Toxoplasmosis, Foot and Mouth Disease- in Some Villages of Baghdad

This study is to investigate Brucellosis, Toxoplasmosis and Foot and Mouth disease of buffalo herds in Baghdad villages. 114 samples (serum) were collected, including 22 for calves less than 6 months and 92 samples of females aged between three to seven years, the samples were collected by staff of Baghdad Veterinary Hospital / Veterinary Directorate, and has been working in Central Veterinary Laboratory/ Veterinary Directorate.
 The ELISA diagnostic kits are selected for the detection of antibodies of Brucella abortus, Toxoplasmosis and Foot and Mouth disease (Multispecies). 
 The result of the research is the presence of different levels of infection in the same herd
 58.18% of Brucella abortus, 11.40% of Toxoplasmosis and 44.73 % of Foot and Mouth disease (Multispecies).
 The purpose of the study is to investigate the three diseases and their compatibility with the symptoms, although buffalo herds have not been previously vaccinated against the diseases, which proves the existence of different levels of infection in one herd and it adversely affect the development of livestock.

Diagnostic Surveillance, Molecular Epidemiology and Phylogenetic Analyses of Rabies Virus Strains in Mali

Rabies is a highly infectious and vaccine preventable disease of humans and animals which is transmitted by dogs. There has been a breadth of emphasis regarding rabies elimination strategies in Asia and Africa due to highly endemic status of rabies in these states. Diagnostic surveillance system is operated by Central Veterinary Laboratory (CVL) only in the capital city of Mali, and hence other cities are at high risk of rabies due to lack of diagnostic labs and reporting mechanism. Therefore, present study has been designed to evaluate the suitability of diagnostic methods of rabies for characterization of circulating rabies virus (RABV) isolates in Mali. Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR), Rapid Immunochromatographic Diagnostic Test (RIDT) and Fluorescent antibody test (FAT) were performed on these 18 brain samples which were previous submitted to the CVL in 2017. Out of these 18 samples, only 16 (88%) were positive for RABV through FAT. These 16 positive samples were processed through RT-PCR and RIDT tests, and both tests further confirmed 15 (93.75%) and 7 (43.75%) samples positive for RABV. Positive samples generated from RT-PCR were sequenced using Sanger sequencing method at Inqaba Biotec and further subjected to phylogenetic analysis. All the sequences analysed by Blastn shared at least 93.5% nucleotide identity to the rabies nucleoprotein gene thereby confirming RABV infection of dogs in Mali. Phylogenetic analysis revealed that all the sequences belonged to the Africa 2 lineage of which 5 were connected to the sub-lineage H, 4 to the sub-lineage F and 2 to the sub-lineage G. The sensitivity of RIDT was low as compared to FAT, while the genetic characterization of the RABV confirmed previous findings of the circulation of the sub-lineages H, F and G belonging to the Africa 2 lineage in Mali. In conclusion, RT-PCR could be used together with FAT for the detection and genetic characterization of RABV in Mali. Further studies using large number of samples are required to validate the suitability of the new RIDT for the diagnosis of rabies in Mali.

Rabies control in Liberia: Joint efforts towards zero by 30

Despite declaration as a national priority disease, dog rabies remains endemic in Liberia, with surveillance systems and disease control activities still developing. The objective of these initial efforts was to establish animal rabies diagnostics, foster collaboration between all rabies control stakeholders, and develop a short-term action plan with estimated costs for rabies control and elimination in Liberia. Four rabies diagnostic tests, the direct fluorescent antibody (DFA) test, the direct immunohistochemical test (dRIT), the reverse transcriptase polymerase chain reaction (RT-PCR) assay and the rapid immunochromatographic diagnostic test (RIDT), were implemented at the Central Veterinary Laboratory (CVL) in Monrovia between July 2017 and February 2018. Seven samples (n=7) out of eight suspected animals were confirmed positive for rabies lyssavirus, and molecular analyses revealed that all isolates belonged to the Africa 2 lineage, subgroup H. During a comprehensive in-country One Health rabies stakeholder meeting in 2018, a practical workplan, a short-term action plan and an accurately costed mass dog vaccination strategy were developed. Liberia is currently at stage 1.5/5 of the Stepwise Approach towards Rabies Elimination (SARE) tool, which corresponds with countries that are scaling up local-level interventions (e.g. dog vaccination campaigns) to the national level. Overall an estimated 5.3 – 8 million USD invested over 13 years is needed to eliminate rabies in Liberia by 2030. Liberia still has a long road to become free from dog-rabies. However, the dialogue between all relevant stakeholders took place, and disease surveillance considerably improved through implementing rabies diagnosis at the CVL. The joint efforts of diverse national and international stakeholders laid important foundations to achieve the goal of zero dog-mediated human rabies deaths by 2030.

Middle East Respiratory Syndrome-Coronavirus Seropositive Bactrian Camels, Mongolia.

Middle East respiratory syndrome coronavirus (MERS-CoV) is a zoonotic disease that was first identified in humans in 2012 in Saudi Arabia. MERS-CoV causes acute and severe respiratory disease in humans. The mortality rate of MERS in humans is ∼35% and >800 deaths have been reported globally as of August 2020. Dromedary camels are a natural host of the virus and the source of zoonotic human infection. In experimental studies, Bactrian camels are susceptible to MERS-CoV infection similar to dromedary camels; however, neither the virus, viral RNA, nor virus-specific antibodies were detected in Bactrian camel field samples so far. The aim of our study was to survey Mongolian camels for MERS-CoV-specific antibodies. A total of 180 camel sera, collected in 2016 and 2017, were involved in this survey: 17 of 180 sera were seropositive with an initial enzyme-linked immunosorbent assay (ELISA) test performed at the State Central Veterinary Laboratory in Mongolia. These 17 positive sera plus 53 additional negative sera were sent to the Rocky Mountain Laboratories, NIAID/NIH, and tested for the presence of antibodies with a similar ELISA, an indirect immunofluorescence assay (IFA), and a virus neutralization test (VNT). In these additional tests, a total of 21 of 70 sera were positive with ELISA and 10 sera were positive with IFA; however, none was positive in the VNT. Based on these results, we hypothesize that the ELISA/IFA-positive antibodies are (1) non-neutralizing antibodies or (2) directed against a MERS-CoV-like virus circulating in Bactrian camels in Mongolia.

Detection of Plasmid-Mediated Colistin Resistant mcr-1 Gene in Escherichia coli Isolated from Infected Chicken Livers in Nepal.

Simple SummaryThe poultry industry is one of the top agribusinesses in Nepal. However, despite the government’s restriction on the use of antibiotics as growth promotors in animals, the overuse and misuse of antibiotics can be seen all over the country. Such inappropriate use of antibiotics has led to the rise of antibiotic resistance among treatment options for both human and animal pathogens. Several findings suggest the failure of colistin, a polymyxin E antibiotic (once regarded as the last resort drug), in the treatment of human bacterial infections is due to the emergence and spread of the plasmid-mediated colistin resistance gene (mcr-1) among Gram-negative bacterial pathogens. The emergence and rapid transfer of resistant strains in poultry farms are associated with unwanted loss of livestock, economic burden and spread of drug-resistance to other animals, humans and the environment, as well. In this study, we characterized the mcr-1 gene from infected chicken livers, where prevalence was found to be alarmingly high. This study identifies the result of regulatory failures. Therefore, this report provides valuable reference to the policy makers so that a more effective policy can be formulated and implemented to curb the spread of drug-resistant pathogens.Background: Plasmid-mediated resistance to the colistin in poultry is considered as an emerging problem worldwide. While poultry constitutes the major industry in Nepal, there is a paucity of evidence on colistin resistance in Escherichia coli isolates causing natural infections in poultry. This study aimed to explore the prevalence of plasmid-mediated colistin resistance gene, mcr-1 in E. coli isolated from liver samples of dead poultry suspected of E. coli infections. Methods: A total of two hundred and seventy liver samples (227 broilers and 43 layers) from dead poultry suspected of colibacillosis were collected from post-mortem in the Central Veterinary Laboratory (CVL), Kathmandu, between 1 February and 31 July 2019. The specimens were processed to isolate and identify E. coli; an antimicrobial susceptibility test (AST) using disk diffusion method was performed with 12 different antibiotics: Amikacin (30 µg), ampicillin (10 µg), ciprofloxacin (5 µg), chloramphenicol (30 µg), cefoxitin (30 µg), ceftazidime (30 µg), ceftriaxone (30 µg), cotrimoxazole (25 µg), gentamicin (10 µg), imipenem (10 µg), levofloxacin (5 µg) and tetracycline (30 µg). Colistin resistance was determined by agar dilution method and colistin-resistant strains were further screened for plasmid-mediated mcr-1 gene, using conventional polymerase chain reaction (PCR). Results: Out of 270 liver samples, 53.3% (144/270) showed growth of E. coli. The highest number (54%; 109/202) of E. coli isolates was obtained in the liver samples from poultry birds (of both types) aged less than forty days. In AST, 95.1% (137/144) and 82.6% (119/144) of E. coli isolates were resistant against tetracycline and ciprofloxacin, respectively, while 13.2% (19/144) and 25.7% (37/144) isolates were resistant to cefoxitin and imipenem, respectively. In the same assay, 76.4% (110/144) E. coli isolates were multi-drug resistant (MDR). The phenotypic prevalence of colistin resistance was 28.5% (41/144). In the PCR assay, 43.9% (18/41) of colistin-resistant isolates were screened positive for plasmid-mediated mcr-1. Conclusion: The high prevalence of mcr-1 in colistin-resistant E. coli isolates in our study is a cause of concern for the probable coming emergence of colistin resistance in human pathogens, due to horizontal transfer of resistant genes from poultry to human isolates.

Field Postmortem Rabies Rapid Immunochromatographic Diagnostic Test for Resource-Limited Settings with Further Molecular Applications.

Functional rabies surveillance systems are crucial to provide reliable data and increase the political commitment necessary for disease control. To date, animals suspected as rabies-positive must be submitted to a postmortem confirmation using classical or molecular laboratory methods. However, most endemic areas are in low- and middle-income countries where animal rabies diagnosis is restricted to central veterinary laboratories. Poor availability of surveillance infrastructure leads to serious disease underreporting from remote areas. Several diagnostic protocols requiring low technical expertise have been recently developed, providing opportunity to establish rabies diagnosis in decentralized laboratories. We present here a complete protocol for field postmortem diagnosis of animal rabies using a rapid immunochromatographic diagnostic test (RIDT), from brain biopsy sampling to the final interpretation. We complete the protocol by describing a further use of the device for molecular analysis and viral genotyping. RIDT easily detects rabies virus and other lyssaviruses in brain samples. The principle of such tests is simple: brain material is applied on a test strip where gold conjugated antibodies bind specifically to rabies antigens. The antigen-antibody complexes bind further to fixed antibodies on the test line, resulting in a clearly visible purple line. The virus is inactivated in the test strip, but viral RNA can be subsequently extracted. This allows the test strip, rather than the infectious brain sample, to be safely and easily sent to an equipped laboratory for confirmation and molecular typing. Based on a modification of the manufacturer's protocol, we found increased test sensitivity, reaching 98% compared to the gold standard reference method, the direct immunofluorescence antibody test. The advantages of the test are numerous: rapid, easy-to-use, low cost and no requirement for laboratory infrastructure, such as microscopy or cold-chain compliance. RIDTs represent a useful alternative for areas where reference diagnostic methods are not available.

Rabies surveillance-response in Mali in the past 18 years and requirements for the future

ContextRabies is endemic in Mali, but little is known about the distribution of rabies and its surveillance across the country. Documenting the evolution of rabies and the problems related to surveillance is useful to facilitate elimination of human rabies by 2030. MethodData collected at the Central Veterinary Laboratory (LCV) from 1999 to 2017 and through the surveillance system functioning at household, health and veterinary structure levels, as established by the Global Vaccine Alliance funded project on the burden of rabies, between 2016 and 2017 in Bamako and the Sikasso region were used in this study. All data on animals examined for rabies by the direct fluorescence antibody test (DFA) during the specified time period were summarized, and the proportion of rabies positive tested among animals suspected of rabies was estimated. From the number of dogs tested positive for rabies, a cumulative incidence was estimated for the total canine population. The number of positive tested samples was divided by the product of the estimated canine population and number of years (18). In addition, the number of human rabies cases was analyzed to estimate a cumulative incidence. ResultsAmong all animal samples suspected for rabies, 93.2% [95% CI 90.6–95.3] were positive by the DFA test. From the 486 included animal samples, 94.7% [95% CI 92.3–96.5] were domestic dogs with 90.9% [95% CI 87.9–93.3] positive, which stands out as the main reservoir of rabies in Mali. Cats, cattle, sheep and donkeys also tested positive using the DFA test. The cumulative incidence of canine rabies was estimated at 0.371 [95% CI: 0.336–0.408] / 10′000 dogs. The highest incidence was observed in Bamako, the capital of Mali, with a cumulative incidence of 2.242 [95% CI: 2.001–2.504] / 10′000 dogs, followed by Koulikoro with 0.335 [95% CI: 0.258–0.429] / 10′000 dogs. For other areas far from LCV, the cumulative incidence was low, with zero in Kidal. Nationally reported cases of human rabies declined in recent years with an average of 12.6 [95% CI: 8.7–16.5] deaths per year. This result gives an annual incidence of 0.1047 [0.0742–0.1352] / 100′000 inhabitants. ConclusionOver the past 18 years, rabies is endemically stable in Mali despite the decrease in reported human cases. The disparity among regions in number of samples tested indicates a low level of clinical and laboratory surveillance and likely a high level of underreporting. Dogs play the main role in rabies transmission in Mali. Monitoring of and decisions for post-exposure prophylaxis (PEP) includes other animals (cats, cattle, and monkeys) involved in transmission. Mali should develop a national rabies strategy to include better communication between the public health and animal health sectors, strengthening of laboratory surveillance capacity, mass vaccination of dogs and guaranteed access to PEP.

Risk factors associated with Avian Influenza subtype H9 outbreaks in poultry farms in Kathmandu valley, Nepal.

The poultry sector contributes four percent to the national GDP of Nepal. However, this sector is under threat with periodic outbreaks of Avian Influenza (AI) subtypes H5 and H9 since 2009. This has been both a public health threat and an economic issue. Since the past few years, outbreaks of AI subtype H9 have caused huge economic losses in major poultry producing areas of Nepal. However, the risk factors associated with these outbreaks have not been assessed. A retrospective case-control study was conducted from April 2018 to May 2019 to understand the risk factors associated with AI subtype H9 outbreaks in Kathmandu valley. Out of 100 farms selected, 50 were “case” farms, confirmed positive to H9 at Central Veterinary Laboratory, Kathmandu, and another 50 farms were “control” farms, matched for farm size and locality within a radius of three km from the case farm. Each farm was visited to collect information using a semi-structured questionnaire. Twelve potential risk factors were included in the questionnaire under the broad categories: birds and farm characteristics, and management and biosecurity status of the farms. Univariable and multivariable logistic regression analysis was conducted and corresponding odds ratios were calculated. Risk factors, associated with AI subtype H9 outbreaks in Kathmandu valley, identified in the final multivariable model were: “farms that have flock size greater than median flock size of study farms (>1500)” (OR = 4.41, 95% CI: 1.53–12.71, p = 0.006), “farms that did not apply rules to wear boots for visitors inside the farms” (OR = 4.32, 95% CI: 1.52–12.29, p = 0.006) and “other commercial farms located within one km periphery” (OR = 10, 95% CI: 1.8–50, p = 0.007). This study showed that outbreaks of AI subtype H9 in Kathmandu valley were associated with a higher population of birds in the farm, poor management practices, and weak biosecurity measures in poultry farms. We suggest improving management practices and increase biosecurity in the farms to reduce incidences of AI subtype H9 outbreaks in Kathmandu valley.