Detection and Molecular Characterization of Parvovirus Serotypes in Egypt

Parvovirus infection is one of the most common enteric diseases affecting dogs in Egypt. A total number of 49 dogs younger than six month old, suffering from severe watery to bloody diarrhea, vomiting and lethargy were examined to confirm the infection with parvovirus through detection of the viral antigen serologically by FASTest® PARVO Card Test and genetically by using specific primers for both conventional and nested polymerase chain reaction (PCR). Serological assay confirmed the infection in 81.63% (40/49) of the examined animals, while the molecular techniques confirmed the infection in 81.63% (40/49) by the conventional PCR and in 97.96% (48/49) by the nested PCR. Sequencing revealed that CPV2a and CPV2b serotypes are circulating in the Egyptian field. The obtained sequences were submitted to the GenBank under the following accession MG764314, MG764315, MG930487 and MG930488. Serotype CPV2b showed some mutation in different sites

Loop-Mediated Isothermal Amplification (LAMP) Assay for Diagnosis of Bovine Babesiosis (Babesia bovis infection) in Egypt

Bovine babesiosis is one of the destructive diseases affecting cattle worldwide especially in tropical and subtropical areas. In Egypt, small livestock holder represents the majority of livestock owners affected by the devastating impact of this disease including costs of diagnosis, treatment, control and prevention as well as limitations of production and reproduction of the affected animals. Early and accurate diagnosis of Babesia spp. infection plays an important role in treatment and control. The current study aimed to evaluate the efficacy of Loop-Mediated Isothermal amplification (LAMP) assay as a new molecular technique used for diagnosis of bovine babesiosis in naturally infected cattle. The confirmation of this infection was depended on blood smears, LAMP and Nested-Polymerase chain Reaction (nPCR) assays, which confirmed the infection in 19%, 47.62% and 52.38% of the examined animals, respectively. Tick samples were collected and identified as Rhipicephalus (Boophilus) annulatus, which is the vector of Babesia spp. Evaluation of blood smears and LAMP assay was carried out against nPCR as a reference test. The obtained results revealed that LAMP assay is a sensitive, specific and cost effective test and will be one of the near future applicable tests in epidemiological and diagnostic studies on babesiosis especially in developing countries endemic with this disease

Loop-Mediated Isothermal Amplification (LAMP) Assay for Diagnosis of Bovine Babesiosis (Babesia bovis infection) in Egypt: LAMP assay for diagnosis of bovine babesiosis

Bovine babesiosis is one of the destructive diseases affecting cattle worldwide especially in tropical and subtropical areas. In Egypt, small livestock holder represents the majority of livestock owners affected by the devastating impact of this disease including costs of diagnosis, treatment, control and prevention as well as limitations of production and reproduction of the affected animals. Early and accurate diagnosis of Babesia spp. infection plays an important role in treatment and control. The current study aimed to evaluate the efficacy of Loop-Mediated Isothermal amplification (LAMP) assay as a new molecular technique used for diagnosis of bovine babesiosis in naturally infected cattle. The confirmation of this infection was depended on blood smears, LAMP and Nested-Polymerase chain Reaction (nPCR) assays, which confirmed the infection in 19%, 47.62% and 52.38% of the examined animals, respectively. Tick samples were collected and identified as Rhipicephalus (Boophilus) annulatus, which is the vector of Babesia spp. Evaluation of blood smears and LAMP assay was carried out against nPCR as a reference test. The obtained results revealed that LAMP assay is a sensitive, specific and cost effective test and will be one of the near future applicable tests in epidemiological and diagnostic studies on babesiosis especially in developing countries endemic with this disease

Detection and Molecular Characterization of Parvovirus Serotypes in Egypt

Parvovirus infection is one of the most common enteric diseases affecting dogs in Egypt. A total number of 49 dogs younger than six month old, suffering from severe watery to bloody diarrhea, vomiting and lethargy were examined to confirm the infection with parvovirus through detection of the viral antigen serologically by FASTest® PARVO Card Test and genetically by using specific primers for both conventional and nested polymerase chain reaction (PCR). Serological assay confirmed the infection in 81.63% (40/49) of the examined animals, while the molecular techniques confirmed the infection in 81.63% (40/49) by the conventional PCR and in 97.96% (48/49) by the nested PCR. Sequencing revealed that CPV2a and CPV2b serotypes are circulating in the Egyptian field. The obtained sequences were submitted to the GenBank under the following accession MG764314, MG764315, MG930487 and MG930488. Serotype CPV2b showed some mutation in different sites

Prevalence of enterotoxins and other virulence genes of Staphylococcus aureus caused subclinical mastitis in dairy cows

Background and Aim: Milk production is one of the main props for the national economy. One of the crucial problems in this industry is subclinical mastitis, which harms this industry that considered the backbone of the economy. It is an infectious and zoonotic disease; the infection can spread between dairy animals through milkers' hands, and milking machines, while the human infection occurs due to the consumption of apparently hygienic milk. Staphylococcus aureus is one of the main causative agents of clinical and subclinical mastitis. It is also considered one of the bacteria incriminated in food intoxication of humans due to its virulence factors as enterotoxins and toxic shock syndrome. The current study was designed to assess the prevalence of S. aureus and its enterotoxins, as well as, its other virulence factors in milk collected from cows that suffer from subclinical mastitis. Materials and Methods: Sixty cows were collected from different dairy farms located in Assiut Governorate, Egypt. These cows were subjected to the clinical examination of the udder and its lymph nodes before sampling. Milk samples were collected from clinically healthy udders. All the milk samples were examined by California mastitis test (CMT), polymerase chain reaction (PCR), and enzyme-linked immunosorbent assay (ELISA) for confirmation subclinical mastitis, presence of S. aureus and its enterotoxins genes and other virulence factors in the examined milk samples. Results: The cows included in the current study had healthy udders. The sixty collected milk samples were tested by CMT. 48/60 (80.0%) were positive samples; from the 48 positive samples, 46 (95.83%) samples were confirmed positive by S. aureus 16s rRNA PCR assay. Multiplex PCRs confirmed the presence of staphylococcus enterotoxin gene C (sec) in one sample, staphylococcus enterotoxin gene D (sed) in 23 samples, while ELISA assay confirmed the presence of the same enterotoxin in only two samples. On the other hand, other groups of genes responsible for some other virulence factors of S. aureus like the extracellular thermostable nuclease (nuc) gene were found in 33 samples, while toxic shock syndrome (tsst) gene and methicillin restraint S. aureus (mecA) gene were not detected in this study. Conclusion: Subclinical mastitis is one of the hidden factors that adversely affect the health of both animals and humans. The milk is usually appeared good and may be consumed by humans especially children; however, it causes severe public health problems. In addition, the infected animals with this form of mastitis can spread the infection to other dairy animals and may be turned to a clinical case of contagious mastitis that may be ended by animal culling or death. S. aureus is one of the main causes of subclinical mastitis in cattle. In addition to extracellular thermostable nuclease (nuc) gene, staphylococcus enterotoxin gene C (sec) and staphylococcus enterotoxin gene D (sed) are the most common virulence genes confirmed in subclinical mastitis milk. These results highlighted the need to apply more hygienic measures in the dairy farms to avoid spreading the infection between animals to ensure the production of safe and healthy food to humans

Assessment of the First Commercial ELISA Kit for the Diagnosis of Theileria annulata

The present study assesses the efficacy of SVANOVIR Theileria annulata-Ab, the first commercial ELISA kit for the diagnosis of Theileria annulata infection in cattle based on a recombinant protein known as T. annulata surface protein (TaSp). As a reference test, a polymerase chain reaction (PCR) assay depending on T. annulata merozoite surface antigen (Tams-1) was applied. A total of 468 blood samples as well as serum samples were randomly collected from cattle and tested in the PCR as well as in the ELISA developed in this study. Moreover, all samples were also analyzed by conventional Giemsa-stained blood smear. The results of this study revealed a good correlation between the results obtained by PCR and the ELISA, whereas all PCR positive samples scored correctly positive in the ELISA and 73 of the 125 PCR negative samples scored correctly negative. Taken together, a sensitivity of 91.25% and a specificity of 78.4% were recorded, when compared to the PCR data. In conclusion, the SVANOVIR Theileria annulata-Ab is a suitable diagnostic assay for use in the diagnosis and epidemiological surveys of Theileria annulata infection in chronic and carrier animals

Assessment of the First Commercial ELISA Kit for the Diagnosis of Theileria annulata

The present study assesses the efficacy of SVANOVIR Theileria annulata-Ab, the first commercial ELISA kit for the diagnosis of Theileria annulata infection in cattle based on a recombinant protein known as T. annulata surface protein (TaSp). As a reference test, a polymerase chain reaction (PCR) assay depending on T. annulata merozoite surface antigen (Tams-1) was applied. A total of 468 blood samples as well as serum samples were randomly collected from cattle and tested in the PCR as well as in the ELISA developed in this study. Moreover, all samples were also analyzed by conventional Giemsa-stained blood smear. The results of this study revealed a good correlation between the results obtained by PCR and the ELISA, whereas all PCR positive samples scored correctly positive in the ELISA and 73 of the 125 PCR negative samples scored correctly negative. Taken together, a sensitivity of 91.25% and a specificity of 78.4% were recorded, when compared to the PCR data. In conclusion, the SVANOVIR Theileria annulataAb is a suitable diagnostic assay for use in the diagnosis and epidemiological surveys of Theileria annulata infection in chronic and carrier animals

Excretion Dynamics of Arboviruses in Mosquitoes and the Potential Use in Vector Competence Studies and Arbovirus Surveillance

The increasing threat of arboviruses such as West Nile virus (WNV) and Usutu virus (USUV) requires the fast and efficient surveillance of these viruses. The examination of mosquitoes takes up an important part; however, these investigations are usually very time-consuming. An alternative sample type for arbovirus surveillance might be mosquito excreta. In order to determine the excretion dynamics under laboratory conditions, laboratory colonies of Aedes vexans and Culex pipiens biotype molestus were infected with WNV, USUV or tick-borne encephalitis virus (TBEV). After infection, the excreta were sampled and investigated for viral RNA. Excretion of viral RNA together with infectious blood meal could be detected up to five days after infection. Further excretion seemed to correlate with a disseminated infection in mosquitoes, at least after USUV infection. In addition, it could be determined that the amount of viral RNA in the excretions correlated positively with the viral load in the mosquito bodies. Overall, this study shows that the usage of mosquito excreta as a sample type for surveillance enables the detection of endemic viruses (WNV, USUV) as well as non-mosquito-borne viruses (TBEV). In addition, examination of viral shedding during vector competence studies can provide insights into the course of infection without sacrificing animals

Vector Competence of German Aedes punctor (Kirby, 1837) for West Nile Virus Lineages 1 and 2

West Nile virus (WNV) is a zoonotic flavivirus transmitted by mosquitoes as a biological vector. Because of its biting behavior, the widespread snow-melt mosquito Aedes punctor could be a potential bridge vector for WNV to humans and nonhuman mammals. However, little is known on its role in transmission of WNV. The aim of this study was to determine the vector competence of German Ae. punctor for WNV lineages 1 and 2. Field-collected larvae and pupae were reared to adults and offered infectious blood containing either an Italian WNV lineage 1 or a German WNV lineage 2 strain via cotton stick feeding. Engorged females were incubated for 14/15 or 21 days at 18 °C. After incubation; surviving mosquitoes were dissected and forced to salivate. Mosquito bodies with abdomens, thoraces and heads, legs plus wings and saliva samples were investigated for WNV RNA by RT-qPCR. Altogether, 2/70 (2.86%) and 5/85 (5.88%) mosquito bodies were found infected with WNV lineage 1 or 2, respectively. In two mosquitoes, viral RNA was also detected in legs and wings. No saliva sample contained viral RNA. Based on these results, we conclude that Ae. punctor does not play an important role in WNV transmission in Germany

Phylogenetic study of Theileria ovis and Theileria lestoquardi in sheep from Egypt: Molecular evidence and genetic characterization

Background and Aim: Ovine theileriosis caused by Theileria ovis and Theileria lestoquardi is an important infectious disease affecting small ruminants in regions of the tropic and subtropic zones. There is limited studies about ovine theileriosis in Egypt; so the present study aims to assess the occurrence of ovine theileriosis in Egypt at the molecular level. Materials and Methods: Blood samples were collected from 115 randomly selected sheep, which were apparently healthy; the ages of the sampled sheep ranged from 1 to 5 years old, from a local breed (barkae and balade), and showed no symptoms indicating infection with Theileria spp. The study was conducted in three governorates representing Lower Egypt (Menoufia and Beheira) and Upper Egypt (El-Wady El-Geded). All blood samples were subjected to polymerase chain reaction (PCR) and semi-nested PCR to target Theileria spp. 18S rRNA genes. Positive samples were sequenced, and these sequences were analyzed using nucleotide basic local alignment search tool (BLAST). Results: Six animals (5.22%) were PCR-positive carriers for ovine theileriosis. Nucleotide BLAST and phylogenetic analyses of the six obtained sequences showed that T. ovis was present in five animals (4.37%) in Menoufia (n=2) and El-Wady El-Geded (n=3), whereas T. lestoquardi was detected in 1 animal (0.87%) in El-Wady El-Geded. Conclusion: This study is the first to provide molecular evidence, genetic characterization, and phylogenetic analysis of ovine Theileria spp. in Egypt. Specifically, T. lestoquardi and T. ovis carrier statuses of sheep were confirmed. These results highlight the importance of developing an effective control strategy against ovine theileriosis carriers that might develop and/or spread theileriosis