Morphological and molecular characterization of Haemonchus contortus isolated from the small ruminants of south Gujarat, India.

The successful design of strategic control measures against the blood-sucking gastrointestinal nematode, Haemonchus contortus in small ruminants can be facilitated by revealing its general features from morphology to the molecular level. In the south Gujarat region of India, a total of 2408 H. contortus were collected from 84 slaughtered sheep's abomasum, consisting of 347 males and 2061 females (1:6 ratio) (p<0.05). Furthermore, 726 H. contortus were collected from 61 goats, comprising 145 males and 581 females (1:4 ratio) (p<0.05). The male worms were approximately 12±0.06 mm long, while female worms were about 20±0.09 mm long. The vulvar morphotypes of the female worms were found to be 17.7% linguiform, 76.6 % knobbed/button (p<0.05), and 5.7 % smooth type, demonstrating common features of H. contortus. The nucleotide sequences of the Internal Transcribed Spacer 1 (ITS-1) of 165 bp or ITS-2 plus of 256 bp were aligned, and it was found that the genotypes of male and female specimens of either sheep or goat origin were identical, with a 100 % match. The present isolates shared >95 % and >94 % homology with published sequences of ITS-1 and ITS-2 plus of H. contortus, respectively, with more nucleotide transitions than transversions in the aligned sequences. The reconstructed phylogram of either ITS-1 or ITS-2 plus revealed two major clades, one for H. contortus and another for other nematodes, with Haemonchus placei showing its proximity with the clade of H. contortus. The study established the role of morphological and molecular features in identifying and differentiating H. contortus parasite at the local level.

Loop-mediated isothermal amplification assay for the rapid detection of Anaplasma marginale in cattle based on major surface protein 5 gene.

BACKGROUND & OBJECTIVES: Timely intervention is needed to minimize the economic losses of vector-borne bovine anaplasmosis which can be possible by the isothermal amplification assay. METHODS: Anaplasma marginale in the cattle of south Gujarat, India was detected in the PCR and LAMP by amplifying the fragment of msp5 gene. The PCR product was digested with EcoRI, and sequenced to confirm its pathogen specific detection. RESULTS: Species specific PCR observed a band of 457 bp of msp5 DNA following 1% agarose gel electrophoresis. Positive LAMP reaction turned into yellow colour while negative sample depicted original pink colour. A detection limit of PCR and LAMP was up to 10-6 and 10-8 of the original genomic DNA of A. marginale, respectively. A single cut site of EcoRI was observed in the PCR product. Current msp5 DNA sequences of A. marginale (MW538962 and MW538961) showed 100% homology with the published sequences. Monophyletic lineage type relationship was observed with high bootstrap proportion among the msp5 DNA sequences of A. marginale in the phylogram. Prevalence rate of A. marginale was significantly higher (p<0.05) in the PCR [43/280 (15.36%)] and LAMP [62/280 (22.14%)] than the microscopic technique [17/280 (6.07%)]. Diagnostic sensitivity, specificity, positive and negative predictive values at 95% CI for LAMP assay with respect to PCR were 93.02%, 90.72%, 64.52% and 98.62%, respectively. INTERPRETATION & CONCLUSION: Thus LAMP can be a practical alternative to the PCR for the diagnosis of A. marginale infection in the cattle even in field condition.

Isolation, identification, and characterization of Listeria spp. from various animal origin foods.

AIM: The present study was undertaken with the prime objective of isolating and identifying Listeria spp. from various foods of animal origin sold at retail market outlets in the city of Navsari, Gujarat. MATERIALS AND METHODS: Total 200 samples comprising of milk, milk products, meat, and fish (50 each) collected aseptically from local market which were subjected first to pre-enrichment in half strength Fraser broth followed by enrichment in full strength Fraser broth and subsequent plating on PALCAM agar. The growth with the typical colony characteristics were further identified up to species level on the basis of their morphological and biochemical characteristics. Cultures identified as Listeria monocytogenes were further subjected to in vitro pathogenicity tests and detection of different virulence-associated genes viz. actA, hlyA, and iap using polymerase chain reaction. RESULTS: Of the total 200 food samples of animal origin; 18 (9%) were found positive for Listeria spp. which were identified as Listeria seeligeri (6, 33.3%), Listeria innocua (5, 27.7%), Listeria welshimeri (4, 22.2%), and L. monocytogenes (3, 16.6%). The highest prevalence was observed in milk samples (8). Species wise, 6 isolates of L. seeligeri which included two each from cow milk, buffalo milk, and meat samples; 5 L. innocua isolates included four recovered from fish and one from meat sample; 4 L. welshimeri comprised of two isolates from ice cream and one each from buffalo milk and meat sample; and 3 isolates of L. monocytogenes recovered from milk (1 cow and 2 buffalo milk). All 3 L. monocytogenes isolates screened for the presence of virulence genes viz. actA, hlyA, and iap using the specific primers revealed the presence of all the genes suggesting the possibility of danger of foodborne listeriosis among raw milk consumers. CONCLUSION: Listeria spp. was isolated from 9% (18/200) of the animal origin food samples viz.; milk, milk products, meat, and fish with the highest prevalence in the milk samples. L. monocytogenes was isolated from 3 milk samples only. L. seeligeri was the predominant species isolated followed by L. innocua, L. welshimeri, and L. monocytogenes in this study. L. monocytogenes were found to carry virulence genes like actA, hly A, and iap genes suggesting the pathogenic potential of these isolates.

Diagnosis of peste des petits ruminants infection in small ruminants through in-house developed Indirect ELISA: Practical considerations.

AIM: The work was conducted to diagnose peste des petits ruminants (PPR) outbreak through an in house developed indirect ELISA (thereafter referred as iELISA) its comparison with other available diagnostic tests and description of practical considerations in its development, utility and limitations. MATERIALS AND METHODS: An outbreak resembled to PPR occurred in two different places of southern Gujarat viz. Vapi and Navsari, affecting 622 animals, including both goat (n = 476) and sheep (n = 146). Animals displayed the typical signs of PPR at Vapi; however diarrhea was the inconsistent feature in animals of Navsari. The affection caused morbidity of 100% and mortality were 73.68% (n = 392/532) and 56.67% (n = 51/90) in Vapi and Navsari outbreaks, respectively. Relevant ante mortem and post mortem samples were collected from representative animals. At the outset of the epidemic no kit was available with us, so agar gel immunodiffusion (AGID) was carried out and a commercial ELISA (cELISA) kit was ordered for making diagnosis through antibody demonstration. Meanwhile, an iELISA was developed in house using PPR vaccine as antigen and protein G conjugated HRPO antibody as detector. Histopathology and results of sandwich ELISA were also used to diagnose PPR virus (PPRV) in the outbreak. RESULTS: The iELISA developed had detected PPRV antibodies in 22/24 samples (91.66%). Significant difference was observed in disease sensitivity pattern of two species by Chi-square test. While AGID failed to detect antibodies in any sample. Results were reconfirmed by comparing with commercially available cELISA kit. CONCLUSION: PPR is an economically important disease and for the rapid diagnosis of PPR the in house developed antibody capture iELISA can be a suitable cost effective alternative.

Characterization of 132 bp Repeats BamH1-H Region in Pathogenic Marek's Disease Virus of Poultry in Gujarat, India, Using PCR and Sequencing.

A total of 34 clinical samples and four Marek's disease virus (MDV) vaccines were tested using primer BamH1/BamH2 in layer birds of poultry. Out of 34 samples tested for detection of MDV, 32 samples produced approximately 434 bp product. All the three HVT vaccines as well as SB-1 (MDV-2) vaccine failed to produce the expected amplicons, there by proving negative for the targeted 132 bp repeats of MDV genome by the primers BamH1/BamH2. Resultant PCR products of the field samples were purified and sequenced and resulted in 378 bp long sequences. PCR was found very satisfactory in detecting the presence of MDV either in feather follicle or in tissue samples. Sequencing study has proved beyond doubt that the two representative samples contained two 132 bp repeats indicating the virulent nature of the field virus.