External quality assessment of Rift Valley fever diagnosis in countries at risk of the disease: African, Indian Ocean and Middle-East regions.

Rift Valley fever virus (RVFV), an arbovirus belonging to the Phlebovirus genus of the Phenuiviridae family, causes the zoonotic and mosquito-borne RVF. The virus, which primarily affects livestock (ruminants and camels) and humans, is at the origin of recent major outbreaks across the African continent (Mauritania, Libya, Sudan), and in the South-Western Indian Ocean (SWIO) islands (Mayotte). In order to be better prepared for upcoming outbreaks, to predict its introduction in RVFV unscathed countries, and to run efficient surveillance programmes, the priority is harmonising and improving the diagnostic capacity of endemic countries and/or countries considered to be at risk of RVF. A serological inter-laboratory proficiency test (PT) was implemented to assess the capacity of veterinary laboratories to detect antibodies against RVFV. A total of 18 laboratories in 13 countries in the Middle East, North Africa, South Africa, and the Indian Ocean participated in the initiative. Two commercial kits and two in-house serological assays for the detection of RVFV specific IgG antibodies were tested. Sixteen of the 18 participating laboratories (88.9%) used commercial kits, the analytical performance of test sensitivity and specificity based on the seroneutralisation test considered as the reference was 100%. The results obtained by the laboratories which used the in-house assay were correct in only one of the two criteria (either sensitivity or specificity). In conclusion, most of the laboratories performed well in detecting RVFV specific IgG antibodies and can therefore be considered to be prepared. Three laboratories in three countries need to improve their detection capacities. Our study demonstrates the importance of conducting regular proficiency tests to evaluate the level of preparedness of countries and of building a network of competent laboratories in terms of laboratory diagnosis to better face future emerging diseases in emergency conditions.

Crystal structure, Hirshfeld surface analysis and inter-action energy, DFT and anti-bacterial activity studies of (Z)-4-hexyl-2-(4-methyl-benzyl-idene)-2H-benzo[b][1,4]thia-zin-3(4H)-one.

The title compound, C22H25NOS, consists of methyl-benzyl-idene and benzo-thia-zine units linked to a hexyl moiety, where the thia-zine ring adopts a screw-boat conformation. In the crystal, inversion dimers are formed by weak C-HMthn⋯OBnzthz hydrogen bonds and are linked into chains extending along the a-axis direction by weak C-HBnz⋯OBnzthz (Bnz = benzene, Bnzthz = benzo-thia-zine and Mthn = methine) hydrogen bonds. A Hirshfeld surface analysis of the crystal structure indicates that the most important contributions for the crystal packing are from H⋯H (59.2%) and H⋯C/C⋯H (27.9%) inter-actions. Hydrogen bonding and van der Waals inter-actions are the dominant inter-actions in the crystal packing. Computational chemistry indicates that in the crystal, the C-HBnz⋯OBnzthz and C-HMthn⋯OBnzthz hydrogen-bond energies are 75.3 and 56.5 kJ mol-1, respectively. Density functional theory (DFT) optimized structures at the B3LYP/ 6-311 G(d,p) level are compared with the experimentally determined mol-ecular structure in the solid state. The HOMO-LUMO behaviour was elucidated to determine the energy gap. Moreover, the anti-bacterial activity of the title compound was evaluated against gram-positive and gram-negative bacteria.

Crystal structure, Hirshfeld surface analysis and inter-action energy, DFT and anti-bacterial activity studies of ethyl 2-[(2Z)-2-(2-chloro-benzyl-idene)-3-oxo-3,4-di-hydro-2H-1,4-benzo-thia-zin-4-yl]acetate.

The title compound, C19H16ClNO3S, consists of chloro-phenyl methyl-idene and di-hydro-benzo-thia-zine units linked to an acetate moiety, where the thia-zine ring adopts a screw-boat conformation. In the crystal, two sets of weak C-HPh⋯ODbt (Ph = phenyl and Dbt = di-hydro-benzo-thia-zine) hydrogen bonds form layers of mol-ecules parallel to the bc plane. The layers stack along the a-axis direction with inter-calation of the ester chains. The crystal studied was a two component twin with a refined BASF of 0.34961 (5). The Hirshfeld surface analysis of the crystal structure indicates that the most important contributions to the crystal packing are from H⋯H (37.5%), H⋯C/C⋯H (24.6%) and H⋯O/O⋯H (16.7%) inter-actions. Hydrogen-bonding and van der Waals inter-actions are the dominant inter-actions in the crystal packing. Computational chemistry indicates that in the crystal, C-HPh⋯ODbt hydrogen bond energies are 38.3 and 30.3 kJ mol-1. Density functional theory (DFT) optimized structures at the B3LYP/ 6-311 G(d,p) level are compared with the experimentally determined mol-ecular structure in the solid state. The HOMO-LUMO behaviour was elucidated to determine the energy gap. Moreover, the anti-bacterial activity of the title compound has been evaluated against gram-positive and gram-negative bacteria.

Isolation and identification of group A rotaviruses among neonatal diarrheic calves, Morocco.

BACKGROUND: Group A rotaviruses (RVA) are the main cause of neonatal calve diarrhea (NCD) in Morocco. In this study, we isolated RVA strains from NCD clinical samples in order to support RVA disease control in Morocco. This isolation process constitutes a first step toward vaccine development. METHODS: Thirteen fecal samples were obtained from calves with a single episode of neonate calf diarrhea at three different dairies and two samples were collected from field during a severe NCD outbreak. Diagnosis of RVA infection was based on fecal immune-chromatographic rapid test and further evaluated for their hemagglutination (HA) activity. RVA isolation was carried out on MA104 cells after inoculates were treated with different concentrations of trypsin TPCK. All RVA isolates were confirmed by LSI VetMAX™ Triplex Ruminant Rotavirus & Coronavirus Real-Time PCR kit. G and P typing were determined by direct sequencing of the VP4 and VP7 amplicons. RESULTS: RVA isolation was achieved for nine clinical samples following one or two passages (60 %) and was properly depended on HA activity and trypsin treatment of inoculates. The first sign of CPE detected consisted of increased cell granularity, obscure cell boundaries, cell rounding, and eventual degeneration and detachment of cells. At lower TPCK concentration (3-10 µg/inoculum), no changes at the cellular level were observed, while cells activated with 25-30 µg of trypsin/inoculums, they degenerated and trypsin cytotoxicity was enhanced. Appreciable changes in cell's morphology were detected with optimal trypsin concentration of 15-20 µg trypsin/inoculums. Data from qRT-PCR confirmed that unsuccessful cultivations have No-Ct, and all nine isolates have Ct values ranged between 12.17 and 24.69. Analysis sequencing revealed that field isolates were of G6 P[5] serotype and isolates from the dairy NCD samples were of G10 P[14] serotype. CONCLUSIONS: To our knowledge, this is the first study in Morocco which reports the circulation of G10P[14] in NCD on dairy farms and G6P[5] in the field. Our study constitutes a crucial and a necessary step allowing preventive and veterinary medicine to support RVA disease controls in the country.

Comparison of SYBR green I real-time RT-PCR with conventional agarose gel-based RT-PCR for the diagnosis of infectious bronchitis virus infection in chickens in Morocco.

BACKGROUND: A rapid, sensitive, and specific molecular method for the diagnosis of infectious bronchitis virus (IBV) infection is important in curbing infectious bronchitis outbreaks in Morocco and other countries. METHODS: In this study, an easy-to-perform SYBR green I real-time reverse transcriptase polymerase chain reaction (RT-PCR) targeting the nucleocapsid gene of IBV was developed and compared with conventional agarose gel-based RT-PCR for the detection of IBV infection. RESULTS: We found that the SYBR green I real-time RT-PCR was at least 10 times more sensitive than the agarose gel electrophoresis detection method. The assay exhibited high specificity for IBV infection. All negative controls, such as Newcastle disease virus, infectious bursal disease virus, and avian influenza virus, were not detected. CONCLUSION: The SYBR green I real-time RT-PCR test described herein can be used to rapidly distinguish IBV from other respiratory pathogens, which is important for diagnosis and control of infectious bronchitis outbreaks in Morocco. The test is a valuable and useful method as a routine assay for diagnosis of clinical IBV infection in commercial chickens.

Molecular epidemiology of canine parvovirus in Morocco.

Since it first emergence in the mid-1970's, canine parvovirus 2 (CPV-2) has evolved giving rise to new antigenic variants termed CPV-2a, CPV-2b and CPV-2c, which have completely replaced the original strain and had been variously distributed worldwide. In Africa limited data are available on epidemiological prevalence of these new types. Hence, the aim of the present study was to determine circulating variants in Morocco. Through TaqMan-based real-time PCR assay, 91 samples, collected from symptomatic dogs originating from various cities between 2011 and 2015, were diagnosed. Positive specimens were characterised by means of minor groove binder (MGB) probe PCR. The results showed that all samples but one (98.9%) were CPV positive, of which 1 (1.1%) was characterised as CPV-2a, 43 (47.7%) as CPV-2b and 39 (43.3%) as CPV-2c. Interestingly, a co-infection with CPV-2b and CPV-2c was detected in 4 (4.4%) samples and 3 (3.3%) samples were not characterised. Sequencing of the full VP2 gene revealed these 3 uncharacterised strains as CPV-2c, displaying a change G4068A responsible for the replacement of aspartic acid with asparagine at residue 427, impacting the MGB probe binding. In this work we provide a better understanding of the current status of prevailing CPV strains in northern Africa.

Prevalence and molecular characterization of avian infectious bronchitis virus in poultry flocks in Morocco from 2010 to 2014 and first detection of Italy 02 in Africa.

The aim of this study was to investigate the prevalence and diversity of infectious bronchitis virus (IBV) genotypes in poultry flocks in 16 areas of Morocco between 2010 and 2014. A total of 360 chicken flocks suspected of being infected by IBV were screened for the IBV N gene using real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Flocks were classified into four groups according to their IBV vaccination programme. Group 1 contained unvaccinated birds. Group 2 received a single application of live H120 vaccine. Groups 3 and 4 birds received one or two booster vaccination(s), respectively, mostly using the H120 vaccine. The real-time RT-PCR results showed that 51.7% of the flocks were positive for the IBV genome with geographical disparities. Molecular characterization of IBV was performed on 50 RT-PCR positive samples by partially sequencing the S1 gene, including the hypervariable regions (nucleotides 705-1097). Two predominant genotypes were detected, with the Massachusetts type dominating (66%), among which 25% of the samples were identical to the H120 vaccine. The second most common genotype (present in 32% of the flocks) was surprisingly Italy 02, revealing the first detection of this genotype in Morocco and also in Africa. 793B, the predominant genotype in the late 1990s in Morocco, was only detected on one occasion and was identical to the 4/91 vaccine strain. This study highlights the high prevalence of IBV in poultry farms in Morocco and confirms its continuous dynamic changes and evolution.

Experimental infection of alpine goats with a Moroccan strain of peste des petits ruminants virus (PPRV).

Peste des petits ruminants virus (PPRV) recently caused a serious outbreak of disease in Moroccan sheep and goats. Alpine goats were highly susceptible to PPRV with mortality rates approaching 100%, as opposed to local breeds of sheep which were less susceptible to the disease. The relative susceptibility of alpine goats was investigated through an experimental infection study with the Moroccan strain of PPRV. Severe clinical signs were observed in the alpine goats with virus being excreted through ocular, nasal and oral routes. No difference in the severity of the disease in goats was observed with different inoculation routes and transmission of the virus by direct contact was confirmed. This study confirmed the susceptibility of the alpine goat to PPRV infection and describes a challenge protocol that effectively and consistently reproduced severe clinical signs of PPR in experimentally infected goats.