Molecular detection and genetic characterization of Coxiella-like endosymbionts in dogs and ticks infesting dogs in Northeast India.

An epidemiological study was performed to determine the role of dogs and ticks infesting dogs in the transmission of Q fever in humans and animals from April 2019 to March 2020 in the northeastern hill states of India. In total, 245 pet and stray dogs irrespective of age or sex were sampled, without specific inclusion or exclusion criteria. In total, 478 ticks belonging to three species were detected, namely Rhipicephalus sanguineus, Rhipicephalus (Boophilus) microplus and Hyalomma anatolicum anatolicum. The DNA extracted from blood and tick samples was assayed for molecular characterization of Coxiella burnetii targeting the 16S rRNA and superoxide dismutase (SOD) genes. Amplified PCR products were purified, cloned and custom sequenced. PCR assay showed 3.3% (8/245) of the dogs were positive for Coxiella-like bacteria. Coxiella-like bacterial DNA was detected in adult fully engorged females of R. sanguineus (7.7%, 13/168), R. (B.) microplus (3.3%, 4/123) and H. anatolicum (1.9%, 1/54). Coxiella-like bacterial DNA lacked in adult male or nymphal stage. The infection rate did not vary significantly between seasons, nor according to sex or age of the host. Six nucleotide sequences of 16S rRNA and SOD genes are discussed.

A 5-Year Prospective Study on Incidence and Clinico-pathological Changes Associated with Naturally Occurring Trypanosomosis in Dogs of Mizoram, India.

PURPOSE: The present research was taken to study the hospital-based incidence and clinico-pathological changes associated with naturally occurring trypanosomosis in dogs of Mizoram. METHODS: A 5-year prospective study on hospital-based incidence and clinico-pathological changes associated with naturally occurring trypanosomosis in dogs of Mizoram was carried out during the study period from April, 2015 to March, 2020. Trypanosoma evansi infection was confirmed by microscopic examination and polymerase chain reaction (PCR). Non-infected clinically healthy dogs (n = 6) served as control. Blood samples were collected to study the haemogram and serum samples were used for the evaluation of serum biochemical parameters and oxidant-antioxidant parameters. RESULTS: During the study period, an overall incidence of 0.25% was recorded for trypanosomosis in dogs. The most consistent clinical findings noticed were anorexia/inappetence, pyrexia, depression/lethargy, pale mucous membrane, dehydration and lymphadenomegaly. Anaemia, granulocytopenia, lymphocytosis and thrombocytopenia were the major findings noticed in trypanosomosis affected dogs. The profile of vital organ function revealed that the mean values of total protein, albumin and random blood glucose were significantly (P < 0.05) lower, whereas the mean values of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total bilirubin, blood urea nitrogen (BUN) and creatinine were significantly (P < 0.05) higher in dogs affected with trypanosomosis. The mean value of lipid hydroperoxide (LPO) was significantly (P < 0.05) higher, whereas the mean values of glutathione (GSH), superoxide dismutase (SOD) and total antioxidant activity (TAOA) were significantly (P < 0.05) lower in trypanosomosis affected dogs. When total erythrocyte count (TEC) was correlated with LPO (r = - 0.631, P < 0.05), a negative correlation was found, while in case of GSH (r = 0.757, P < 0.05), SOD (r = 0.767, P < 0.05) and TAOA (r = 0.713, P < 0.05), it was positively correlated. CONCLUSION: A negative correlation of TEC count with LPO, while a positive correlation with GSH, SOD and TAOA signify the role of oxidative stress in the pathogenesis of anaemia induced by T. evansi infection in dogs. The present study findings might be helpful to clinicians when treating clinical cases of this kind. Incorporation of organ protective drugs and antioxidants in the treatment schedule may result in better prognosis.

Molecular detection of Babesia microti in laboratory mice from India.

BACKGROUND & OBJECTIVES: For detection and molecular characterization of Babesia microti in laboratory mice from India. METHODS: A total of 625 mice were screened by peripheral blood smear examination and subsequently was confirmed by PCR using a piroplasm conserved primer set (Piro A/B). Nested PCR was done using a species-specific primer targeting the gene encoding the small subunit ribosomal RNA (18S rRNA). The PCR products were cloned, purified and sequenced. A total of 12 isolates were obtained. The sequences were aligned and phylogenetic trees were prepared with other published Babesia spp. sequences. RESULTS: B. microti was detected with a total infection rate of 8.80%. The higher rate of infection was observed by species specific PCR (8.80%) than examined by blood smear (7.20%). Sequence and phylogenetic analysis showed that Babesia species detected in mice were genetically identical to the genotypes of B. microti and can be easily distinguished from other genotypes of Babesia parasites by neighbour joining and maximum likelihood method. Intra-species analysis indicated that all the twelve isolates from six North-Eastern states of India have a close identity but inter-species showed genetic reservoir host for transmission of babesial infection to humans. INTERPRETATION & CONCLUSION: The detection of Babesia microti may suggest that laboratory mice may serve as potential reservoir host for human infection and possibility of innovative way of diagnosing and control of human babesiosis.

Incidence of cutaneous habronemosis in Manipuri ponies in India.

Information pertaining to parasitic fauna and parasitic diseases in Manipuri ponies in India is not available. Moreover, no systematic studies have been undertaken on cutaneous habronemosis in Manipuri ponies which is a common skin problem of Manipuri ponies as reported by pony owners. Keeping in the view of the importance of parasitic infections in veterinary health coverage particularly in Manipuri ponies, the present study was planned. A survey of natural cases of cutaneous habronemosis followed by molecular confirmation of species involved and treatments were done. Out of 200 ponies examined, nine cases (4.5%) of cutaneous habronemosis was recorded. Gross examination revealed raised and ulcerated wounds with necrotic tissues covered with yellowish-tan granulation. Histopathological study revealed eosinophilic granuloma and in the center of the granuloma with necrotic debris. Remnants of the Hebronema larvae with infiltrating neutrophils surrounded by proliferating fibrous tissue with numerous eosinophils, macrophages and lymphocytes were also observed. Molecular detection of Habronema sp. was confirmed by semi-nested PCR. Sequence analysis revealed larvae of H. muscae was the common spirurid species responsible for producing cutaneous habronemosis in Manipuri ponies. Subsequently, sequence submitted to NCBI GenBank and accession number obtained (MH038181). Surgical removal of necrotic tissue, ivermectin injection along with antibiotics successfully cured all the lesions in infected ponies.Results confirmed occurrence of cutaneous habronemosis in Manipuri ponies in India.

Molecular Characterization of Chitin Synthase Gene of Demodex canis from Mizoram, India.

AIMS: Canine demodicosis is a parasitic condition affecting the skin of dogs. The present study was designed to characterize chitin synthase gene of Demodex canis. The molecular technique was used for better understanding of this gene. METHODS: A total of 75 dogs which are reared as pets with or without showing any skin lesions were examined during the study period. Skin scrapings were examined by indirect method using 10% potassium hydroxide solution under 10 × microscope. DNA samples were extracted from positive skin samples and were subjected to PCR for molecular identification. RESULTS: A total of 25 dogs irrespective of age, sex, breed or coat showed positive result for D. canis. The PCR revealed a single amplified product of 339 bp length which exactly matched with D. canis. The chitin synthase gene was amplified by PCR, subsequently cloned, sequenced, and compared with available data in GenBank for the particular gene of D. canis. Only one single nucleotide polymorphism (SNP) was noticed at 231 position of the chitin synthase gene sequence when compared to other isolates. CONCLUSION: The molecular technique confirms with the morphological identity of D. canis. This report signifies the value of peculiar tool to identify 'follicular mite' even from apparently healthy skin.

Quantification and expression studies of IL-13 gene in response to experimental infection with Haemonchus contortus in Garole and Sahabadi sheep.

To evaluate the role of IL-13 gene in Garole/Sahabadi sheep against resistant/susceptibility to Haemonchus contortus five Garole sheep (Group I) with consistently showing low egg per gram of faeces (EPG) (≤150) and another five Garole sheep (Group II) with high EPG (≥500) were selected and fed orally with active H. contortus third stage (L3) larvae at the dose rate of 500 larvae/kg body weight. Five Sahabadi sheep (Gr-III) known susceptible to H. contortus were also challenged with the same dose as the previous groups. Blood was collected from each group on day 0, 7 and 14days post infection (dpi). The serum level of IL-13 cytokine was evaluated by ELISA at different days of post infection. The ovine IL-13 was partially isolated and the RNAm level of IL-13 expression was studied by real-time PCR at different days post infection. In the resistant Garole sheep (Gr-I) the concentration of IL-13 cytokine increased significantly (P≤0.05) 14days PI compared to susceptible Garole (Gr-II)/Sahabadi (Group-III). In similar way the relative expression of IL-13 in group I (Gr-I) was found more than seven fold higher than Gr-II and Gr-III on 14 dpi. The Th2 type immune response to H. contortus characterized by higher serum level of IL-13 cytokine and IL-13 gene expression indicated that IL-13 gene plays a vital role for the reduced establishment and/or survival of established H. contortus in resistant Garole sheep.

Occult Dirofilariosis in Dogs of North Eastern Region in India.

BACKGROUND: The North Eastern Region in India is endemic for canine heartworm disease but in clinics accurate diagnosis is some times difficult. The aim of the present study was to determine the prevalence of occult infections for heartworm disease in canine in two geographical regions of North Eastern India. METHODS: A total of 782 numbers of three categories of dogs namely, working dogs of military and paramilitary forces, pet dogs and stray dogs were screened for the presence of heartworm infection from August 2011 to July, 2012 in Guwahati (Assam) and Aizawl (Mizoram). Conventional, immunological and molecular techniques were followed for this epidemiological study. The criteria to determine the occult heartworm cases were based on the differences between heartworm positive cases in PCR test and antigen ELISA test. RESULTS: The findings revealed an overall 22.69 percent occult case. The working dogs had highest prevalence (60%) followed by pet (29.16%) and stray dogs (17.75%). CONCLUSION: The highest percentage of occult heartworm infection was present in working dogs maintained under military or paramilitary forces.

Prevalence and molecular epidemiological data on Dirofilaria immitis in dogs from Northeastern States of India.

The aim of the present study was to determine the prevalence of Dirofilaria immitis in stray, pet, and working dogs (n = 413, 266, and 103, resp.) from Guwahati (Assam) and Aizawl (Mizoram), areas located in two Northeastern States of India. Diagnostic methods applied were microscopy (wet film and Knott's concentration technique), immunological test (Ag ELISA by SNAP 4Dx ELISA kit), and molecular tools (polymerase chain reaction and sequencing), which evidenced 11.38, 18.03, and 13.93% of positive animals, respectively. No significant differences were observed by area (18.23% versus 17.68%) nor by sex (18.1% versus 17.9%), whereas stray dogs proved more infected than other groups (P < 0.05). ELISA test evidenced an overall 22.69% of occult infections, mainly in working dogs (60%), and molecular techniques detected Dirofilaria (Nochtiella) repens in 4 stray dogs from Guwahati. Characterization of D. immitis isolates for ITS-2 region showed close identity with South Asian isolates.