Molecular epidemiology and associated risk factors of Anaplasma marginale in camels and possible co-infections.

Anaplasma spp. is an important pathogen that affects a wide range of animals, including camels. The current study aimed to assess the prevalence of six Anaplasma spp. in 400 camels from Ismailia, Suez, and Sharkia governorates in northern Egypt, as well as their associated risk factors and possible coinfections. Blood and fecal samples were examined using bacterial culture, the vitek2 system, and PCR. Genetic divergence among Anaplasma marginale (A. marginale) isolates was characterized using the msp4 gene. The overall prevalence of A. marginale was 19.5%. Sequencing analysis confirmed the PCR results, and a single A. marginale genotype was recognized by msp4 sequencing. The phylogenetic tree indicated that the study A. marginale isolates clustered together and were close to Egyptian A. marginale identified from buffalo (OP142725 and OP142726). Age, sex, housing type, tick infestation, body conditions, and tick control factors were significantly associated with camel anaplasmosis using a logistic regression model (odds ratio >1, P < 0.05). Multivariate logistic regression analysis revealed that the infection was 2.03, 1.9, 2.6, 1.9, and 1.8 times higher in females, semi-enclosed housing, ages >5 years, tick infestation, and emaciated camels. The risk of infection due to a tick control factor increased by 4.4 and 2.6 times when no control was applied or with irregular control, respectively. This is the first molecular report of A. marginale infection in camels in Ismailia, Suez, and Sharkia in northern Egypt, indicating a moderate prevalence of A. marginale and the involvement of multiple bacterial infections, mainly Escherichia coli and Salmonella spp. Thus, it is necessary to develop effective management and control for camel anaplasmosis.

Epidemiology and diagnostic accuracy of Clostridium perfringens toxins in the intestinal contents of camels, sheep, and cattle: a cross-sectional study in Dakahlia governorate, Egypt.

This study aimed to establish an accurate epidemiological surveillance tool for the detection of different C. perfringens types from 76 diseased and 34 healthy animals in Dakhalia Governorate, Egypt. A total of 110 intestinal content samples were randomly collected from camels, sheep, and cattle. C. perfringens was isolated and biochemically identified by the VITEK2 system. Toxinotyping and genotyping of C. perfringens isolates were specified by a multiscreen ELISA and real-time qPCR (rt-qPCR). The occurrence of C. perfringens was highest among camels (20% in healthy and 25% in diseased) and was lowest in cattle (23.1% and 14.7%). The cpa toxin was detected in all isolates by rt-qPCR and in 7 isolates by ELISA, ext toxin was detected in 7 isolates by rt-qPCR and in 6 isolates by ELISA, and cpb toxin was detected in 2 isolates by both rt-qPCR and ELISA. Four types of C. perfringens were identified by rt-qPCR, type A (65.2%), B (4.3%), C (4.3%), and D (26.1%), and three types by ELISA, type D (17.4%), A (8.7%) and C (4.3%). Our study indicated the prevalence of infection in Dakahlia by C. perfringens type A and D, particularly camels, and recommends adopting an appropriate vaccination strategy among the studied animals.

Molecular prevalence, associated risk factors and genetic characterization of Trypanosoma evansi in camels.

Surra is a major infectious disease of camels being caused by Trypanosoma evansi (T. evansi) in developing countries, including Egypt. However, the identification of changes in the T. evansi prevalence in Egypt is important. In this study, the prevalence of T. evansi and its associated risk factors as well as the genetic characterization of the parasite were estimated. Blood samples were collected from 163 camels from two governorates in Lower Egypt. PCR targeting RoTat 1.2VSG was used for the detection of T. evansi and internal transcribed spacer 1 (ITS-1) was used for sequencing analysis and genetic characterization. Overall prevalence was 19.6% using RoTat 1.2VSG. The risk of the infection in females was 4 times higher than in males (P = 0.0004, OR = 4; 95% CI = 0.79-8.96) and in camels with a history of clinical signs it was 2.3 times higher than camels without clinical signs (P = 0.04, OR = 2.3, 95% CI = 1.035-5.15). Analysis of the ITS-1 sequences of four T. evansi isolates showed little heterogeneity compared to similar sequences in the database. Sequence and phylogenetic analysis, based on the ITS-1 region, confirmed the presence of two distinct genotypes of T. evansi in Egyptian camels with more than 99% similarity with T. evansi isolates from different countries across the ITS-1 region and were closely related to Filipino and Chinese isolates. The results of the study can be used for the observation and prevention of disease and updating the epidemiological data.

Lumpy skin disease in cattle in Sharkia, Egypt: epidemiological and genetic characterization of the virus.

Lumpy skin disease virus (LSDV) continues to threaten the cattle industry in Egypt. This survey investigated the epidemiological risk factors and the genetic characterization of circulating strains by partial sequencing of the P32 gene on cattle farms in the Sharkia Governorate, Egypt. Out of 600 cattle examined, morbidity, mortality, and case fatality were 31.2%, 1.8%, and 5.9%, respectively. Risk of LSD was higher among unvaccinated cattle kept outdoors compared to vaccinated cattle kept indoors, and the prevalence rates were statistically significantly different (P < 0.05). Regarding seasonal distribution, the highest number of cases was in June and July, and the lowest was in November. The P32 gene sequences showed that two LSDV isolates were 100% identical and 99.26% identical with 2017 Russian LSDV. Phylogenetic analysis revealed that two local isolates in this study were grouped together with other LSDVs from Russia (Saratov), Kenya, Greece, and Israel. The sequences in the study and other Egyptian sequences were grouped into two clusters with low genetic divergence, indicating that different strains are spreading in Egypt and that LSDV is more genetically related to sheep poxviruses than goat poxviruses. Our study confirms the necessity of evaluating the vaccination strategy adopted in Egypt, and sequence analysis based on the P32 gene is appropriate for genetic epidemiological studies of the local LSDVs.

Transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to animals: an updated review.

COVID-19 caused by a novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) originated in Wuhan (Hubei province, China) during late 2019. It has spread across the globe affecting nearly 21 million people with a toll of 0.75 million deaths and restricting the movement of most of the world population during the past 6 months. COVID-19 became the leading health, economic, and humanitarian challenge of the twenty-first century. In addition to the considerable COVID-19 cases, hospitalizations, and deaths in humans, several cases of SARS-CoV-2 infections in animal hosts (dog, cat, tiger, lion, and mink) have been reported. Thus, the concern of pet owners is increasing. Moreover, the dynamics of the disease requires further explanation, mainly concerning the transmission of the virus from humans to animals and vice versa. Therefore, this study aimed to gather information about the reported cases of COVID-19 transmission in animals through a literary review of works published in scientific journals and perform genomic and phylogenetic analyses of SARS-CoV-2 isolated from animal hosts. Although many instances of transmission of the SARS-CoV-2 have been reported, caution and further studies are necessary to avoid the occurrence of maltreatment in animals, and to achieve a better understanding of the dynamics of the disease in the environment, humans, and animals. Future research in the animal-human interface can help formulate and implement preventive measures to combat the further transmission of COVID-19.

Potential efficiency of conventional and advanced approaches used to detect Mycobacterium bovis in cattle.

The present study was aimed to assess the prevalence and efficiency of techniques for the diagnosis of bovine tuberculosis (bTB) including enzyme-linked immunosorbent assay (ELISA), Gamma interferon assay (IFN-γ) and polymerase chain reaction (PCR) in comparison to skin tuberculin test and culture technique. A total of 2600 cross-breed dairy cattle in Menoufia and Daqahlia governorates were tested by the single intradermal tuberculin test where the disease prevalence was 1.8%. Serum and whole blood samples were collected from positive tuberculin reactors for ELISA and IFN-γ assay, respectively. After slaughtering of positive tuberculin reactors, the post-mortem examination was carried out and tissue samples were collected for the bacteriological examination and PCR. The percentage of visible lesions of tuberculin reactors was 78.7%, while non-visible lesions were 21.27%. Culture technique revealed that the percentage of bTB was 63.8%. The ELISA and IFN-γ assay using short-term culture filtrate (ST-CF) prepared antigen revealed higher sensitivity (72.3% and 82.9%) than the bovine purified protein derivative (PPD-B) antigen. Although prepared ST-CF antigen has great efficiency and eligibility for the diagnosis of bTB, PCR appeared to have a higher sensitivity (85.1%) than other diagnostic methods when dealing with post-mortem samples. Gamma interferon assay using ST-CF antigen is recommended for antemortem diagnosis of bTB in cattle.

Molecular survey and characterization of Trypanosoma evansi in naturally infected camels with suspicion of a Trypanozoon infection in horses by molecular detection in Egypt.

In Egypt, although the Trypanosoma evansi has been reported frequently among domestic animals, there is no published data on T. evansi in horses. Therefore, this study aimed to assess the prevalence and characterization of T. evansi in three governorates by examining blood samples from 40 local camels, 35 imported camels, 25 horses and 10 donkeys by PCR targeting the sequences of TBR and RoTat 1.2VSG. The overall prevalence of T. evansi was 54.5% and 21.8% by TBR PCR and RoTat 1.2VSG PCR, respectively. The TBR PCR detected T. evansi in 60% and 71.4%, respectively, of local and imported camels and in 10% and 40% of donkeys and horses, respectively. For RoTat 1.2VSG PCR, T. evansi was detected in 32.5% and 31.4 of local and imported camels, respectively. All horses and donkeys were negative by RoTat 1.2VSG PCR. TBR PCR was superior to RoTat 1.2VSG PCR in T. evansi infection detection. Statistically significant differences in the prevalence of the infection were observed on the basis of body condition and location. Sequencing and phylogenetic analysis, based on RoTat 1.2VSG, confirmed the presence of T. evansi, which was closely related to Egyptian and Indian isolates. In conclusion, TBR PCR is the best assay to monitor T. evansi infections in camels, horses, and donkeys. The presence of T. evansi in horses and donkeys possibly play a role in the transport of the infection to camels. This is the first report of T. evansi infection in horses in Egypt using TBR PCR.

Parasitological, Serological and Molecular Prevalence of Trypanosoma evansi among Arabian Camels (Camelus dromedaries) with Evaluation of Antitrypanosomal Drugs.

PURPOSE: This study was carried out to assess the prevalence of Trypanosoma evansi infection in naturally diseased Dromedary camels in Dammam, Eastern region of Saudi Arabia. The detection of Trypanosoma evansi was performed using the parasitological, serological, and molecular diagnosis and a comparison between such methods were analyzed. In addition, evaluation of therapeutic efficacy of selected antitrypanosomal drugs, cymelarsan and quinapyrmine (aquin-1.5), was trialed for treatment of diagnosed infected cases. METHODS: A total 350 randomly selected camels were evaluated using thin blood smear (TBS), RoTat1.2 PCR and CATT/T. evansi techniques. RESULTS: The total prevalence was 6.9%, 7.7%, and 32.8% by TBS, RoTat1.2 PCR and CATT/T. evansi techniques, respectively. Although PCR detect T. evansi in more samples than TBS, the agreement was good (K = 0.9). Among the CATT/T. evansi results, PCR detect T. evansi in 12 and 15 CATT positive and negative camels, respectively, with low agreement (Kappa = 0.1). The use of cymelarsan and quinapyramine sulfate in the treatment of naturally infected cases demonstrated a very efficient therapeutic response. CONCLUSION: It was found that 1. Comparing the CATT/T. evansi and PCR results, the positivity of CATT was higher than PCR detection, while the agreement was poor (K = 0.1). 2. Cymelarsan and aquin-1.5 proved to be effective in the treatment of naturally infected camels, but cymelarsan presented with higher effectiveness (100%) than aquin-treated camels (83.3%). a 3. The use of cymelarsan and CATT is recommended for disease treatment and control.

Seroprevalence, associated risk factors, and molecular detection of bovine brucellosis in rural areas of Egypt.

The study was carried out on six villages in northern Egypt to evaluate the epidemiological situation of bovine brucellosis among 989 unvaccinated household cattle by Rose Bengal Plate Test (RBPT) and indirect ELISA (iELISA) and to investigate the existence of Brucella DNA using real-time PCR in 100 milk and 100 sera from seropositive cattle and 50 sera from seronegative cattle. The overall seroprevalence was 20.7% and 23.7% by RBPT and iELISA, respectively. Based on the iELISA results, the seroprevalence was significantly (P < 0.05) higher in the village II (34.7%) and cattle > 7 years (30.1%). More males than females were non-significant seropositive (P = 0.6). There was 95% agreement between RBPT and iELISA, although iELISA showed a higher positivity rate (23.7%, 95% CI: 0.21-0.26) than RBPT (20.7%, 95% CI: 0.18-0.24). DNA of Brucella was confirmed in 16 milk samples by IS711 qPCR from seropositive cattle, however, no Brucella DNA was detected in serum samples tested positive and negative. Brucella abortus was the only species detected based on the alkB gene. Prevalence is highly related to the sampling site and the age of the animals. In conclusion, although qPCR is more accurate and commonly used in the diagnosis of most infectious diseases but in this situation iELISA is preferred and recommended for continuous screening and animal movement restriction and vaccination protocols, especially in high-risk areas.

Genes Encoding the Virulence and the Antimicrobial Resistance in Enterotoxigenic and Shiga-Toxigenic E. coli Isolated from Diarrheic Calves.

Calf diarrhea is one of the considerable infectious diseases in calves, which results in tremendous economic losses globally. To determine the prevalence of Shiga-toxigenic E. coli (STEC) and Enterotoxigenic E. coli (ETEC) incriminated in calf diarrhea, with special reference to Shiga- toxins genes (stx1 and stx2) and enterotoxins genes (lt and sta) that govern their pathogenesis, as well as the virulence genes; eaeA (intimin) and f41(fimbrial adhesion), and the screening of their antibiogram and antimicrobial resistance genes; aadB, sul1, and bla-TEM, a total of 274 fecal samples were collected (April 2018-Feb 2019) from diarrheic calves at different farms in El-Sharqia Governorate, Egypt. The bacteriological examination revealed that the prevalence of E. coli in diarrheic calves was 28.8%. The serotyping of the isolated E. coli revealed 7 serogroups; O26, O128, O111, O125, O45, O119 and O91. Furthermore, the Congo red binding test was carried out, where 89.8% of the examined strains (n = 71) were positive. The antibiogram of the isolated strains was investigated; the majority of E. coli serotypes exhibit multidrug resistance (MDR) to four antimicrobial agents; neomycin, gentamycin, streptomycin, and amikacin. Polymerase chain reaction (PCR) was used to detect the prevalence of the virulence genes; stx1, stx2 lt, sta, f41 and eaeA, as well as the antibiotic resistance genes; aadB, sul1, and bla-TEM. The prevalence of STEC was 20.2% (n = 16), while the prevalence of ETEC was 30.4% (n = 24). Briefly, the Shiga toxins genes; stx1 and stx2, are the most prevalent virulence genes associated with STEC, which are responsible for the pathogenesis of the disease and helped by the intimin gene (eaeA). In addition, the lt gene is the most prevalent enterotoxin gene accompanied by the ETEC strains, either alone or in combination with sta and/or f41 genes. The majority of pathogenic E. coli incriminated in calf diarrhea possesses the aadB resistance gene, followed by the sul1 gene. Enrofloxacin, florfenicol, amoxicillin-clavulanic acid, and ampicillin-sulbactam, are the most effective antimicrobial agents against the isolated STEC and ETEC strains.

Prevalence, the antibiogram and the frequency of virulence genes of the most predominant bacterial pathogens incriminated in calf pneumonia.

The purpose of this study was to investigate the prevalence, antibiotic resistance and certain virulence genes of the most predominant bacterial pathogens causing BRD. A total of 225 calves; 55 apparently healthy and 170 diseased; were sampled. Bacteriological examination, antimicrobial susceptibility testing and PCR based detection of some virulence genes were performed. In addition, the serotyping of E. coli was performed using the slide agglutination test. The most predominant bacterial pathogens retrieved from apparently healthy calves were E. coli (16.4%) and S. aureus (10.9%), and in pneumonic calves were E. coli (23.5%), P. vulgaris (12.4%) and S. aureus (11.8%). The most prevalent virulence gene in E. coli was the fimH gene (100%), followed by eaeA gene (24.5%) and hly gene (20.4%). All the examined S. aureus strains harbored spa and coa genes; likewise, all P. multocida strains harbored toxA gene. The majority of the isolated strains displayed remarkable sensitivity to norfloxacin and enrofloxacin; furthermore, the retrieved E. coli strains exhibited multidrug-resistance to gentamicin, erythromycin, streptomycin and trimethoprim-sulphamethoxazole, in addition, the isolated S. aureus and P. aeruginosa strains showed multidrug-resistance to amoxicillin, ampicillin and tetracycline. E. coli serogroups including O18, O143, O1, and O6 were retrieved from pneumonic calves as the first report in Egypt. In conclusion, the synergism between the conventional and genotypic analysis is an effective gadget for the characterization of bacterial pathogens causing BRD. Continuous surveillance of antimicrobial susceptibility is essential to select the drug of choice due to the development of multidrug-resistant strains.

Modeling the potential risk factors of bovine viral diarrhea prevalence in Egypt using univariable and multivariable logistic regression analyses.

AIM: The present cross-sectional study was conducted to determine the seroprevalence and potential risk factors associated with Bovine viral diarrhea virus (BVDV) disease in cattle and buffaloes in Egypt, to model the potential risk factors associated with the disease using logistic regression (LR) models, and to fit the best predictive model for the current data. MATERIALS AND METHODS: A total of 740 blood samples were collected within November 2012-March 2013 from animals aged between 6 months and 3 years. The potential risk factors studied were species, age, sex, and herd location. All serum samples were examined with indirect ELIZA test for antibody detection. Data were analyzed with different statistical approaches such as Chi-square test, odds ratios (OR), univariable, and multivariable LR models. RESULTS: Results revealed a non-significant association between being seropositive with BVDV and all risk factors, except for species of animal. Seroprevalence percentages were 40% and 23% for cattle and buffaloes, respectively. OR for all categories were close to one with the highest OR for cattle relative to buffaloes, which was 2.237. Likelihood ratio tests showed a significant drop of the -2LL from univariable LR to multivariable LR models. CONCLUSION: There was an evidence of high seroprevalence of BVDV among cattle as compared with buffaloes with the possibility of infection in different age groups of animals. In addition, multivariable LR model was proved to provide more information for association and prediction purposes relative to univariable LR models and Chi-square tests if we have more than one predictor.

Lumpy skin disease in cattle: Frequency of occurrence in a dairy farm and a preliminary assessment of its possible impact on Egyptian buffaloes.

Lumpy skin disease (LSD) is an endemic infectious disease of cattle in Egypt. This survey aimed to define the prevalence of clinical and sub-clinical LSD virus (LSDV) infection among cattle and investigate their contact with water buffaloes (Bubalus bubalis) in order to improve the understanding of LSD epidemiology. Cattle and buffalo were examined owing to the appearance of skin lesions. Because clinical signs were consistent with LSDV infection, samples from cattle in a non-grazing dairy farm (n = 450) were submitted for LSDV testing together with those from the in-contact buffaloes (n = 100). Results revealed that the intra-herd percentage of cattle infected with LSDV varied with the detection method. This ranged from 22.4% to 65.4% by virus isolation (VI) and polymerase chain reaction (PCR), respectively, in clinical cattle samples, compared to 0% and 10% by VI and PCR in non-clinical cases. Using the neutralising index (NI), LSDV antibodies were found in 100% (n = 100) of the tested cow's sera (NI = > 2.0 and ≥ 3.0), whereas buffalo's sera (n = 34) displayed little increase in antibody level (NI ≥ 1.5). None of the buffalo were positive for LSDV by VI and PCR. In addition, there were no significant differences in LSD prevalence among the cattle with regard to age and sex. In conclusion, the occurrence of LSD in cattle warrants a further epidemiological study of the spread of the disease in the area and adoption of control and prevention strategies. In addition, the PCR assay was confirmed to be useful in the diagnosis of LSDV and for wider epidemiological studies.

Complete Genome Sequence of Bovine Viral Diarrhea Virus-1 Strain Egy/Ismailia/2014, Subtype 1b.

Here, we report the complete genome sequence of bovine viral diarrhea virus-1b (BVDV-1b), strain Egy/Ismailia/2014. The virus genome is composed of 12,217 nucleotides organized as one open reading frame encoding 3,898 amino acids. This report will assist efforts in diagnostics, studying molecular epidemiology, and control of BVDV in Egypt.

Development of multiplex real-time PCR assay for the detection of Brucella spp., Leptospira spp. and Campylobacter foetus.

Abortion among dairy cattle is one of the major causes of economic losses in the livestock industry. This study describes a 1-step multiplex real-time polymerase chain reaction (PCR) to detect Brucella spp., Leptospira spp. and Campylobacter foetus, these are significant bacteria commonly implicated in bovine abortion. ß-actin was added to the same PCR reaction as an internal control to detect any extraction failure or PCR inhibition. The detection limit of multiplex real-time PCR using purified DNA from cultured organisms was set to 5 fg for Leptospira spp. and C. foetus and to 50 fg for Brucella spp. The multiplex real-time PCR did not produce any non-specific amplification when tested with different strains of the 3 pathogens. This multiplex real-time PCR provides a valuable tool for diagnosis, simultaneous and rapid detection for the 3 pathogens causing abortion in bovine.

Molecular and parasitological detection of Trypanosoma evansi in Camels in Ismailia, Egypt.

Trypanosoma evansi (T. evansi) is an endemic disease of camels and other domestic animals in Egypt. This study aimed to determine the prevalence of clinical and sub-clinical T. evansi infection among camels in Ismailia, Egypt, as well as survey their owners for T. evansi infection. The diagnostic sensitivity of three different PCR assays for detection of T. evansi in blood samples was evaluated. Blood samples were collected from 100 camels and 20 of their owners in the Ismailia governorate. Results revealed that the percentage of infected of camels with T. evansi vary with the detection method, ranging from 10% to 46% by PCR compared to 12% by microscopic examination of stained blood smears. Targeting the highly repeated sequence of mini-chromosome satellite DNA (TBR1/2 primer set) was more often seen in the PCR method (46% positive) compared to targeting ITS 1 (16% positive) or RoTat 1.2 VSG (10% positive) sequences. A partial sequence of RoTat 1.2 VSG gene was identical to the T. evansi sequences reported from India and Kenya, but varied similarity was seen when aligned with Egyptian T. evansi sequences. None of the camel owners were positive for T. evansi by microscopic examination of stained blood smears or PCR assays. PCR assay based on TBR sets is useful in the diagnosis and control disease and reducing economic losses.