Comparison of in-vitro bioassays for evaluation of the response of different stages of Rhipicephalus sanguineus sensu lato to calcium alginate encapsulated pheromone beads.

Tick sex pheromone (SP), assembly pheromone (AP) and their combination (SP + AP) were encapsulated in calcium alginate beads. In vitro bioassays, namely Petri dish and olfactometer assays, were employed to estimate the level of attraction of the various stages of Rhipicephalus sanguineus sensu lato, namely unfed and engorged (fed) larvae, nymphs, females, and males to the different pheromones. The study revealed that only the Petri dish assay was suitable to evaluate the response of larval stages whereas the olfactometer bioassay could also be used for evaluating the response of all other stages. Attraction to pheromone encapsulated calcium alginate beads of all tick stage was higher in the Petri dish assay than in the olfactometer assay.

Hypodermosis in cattle translocated to Tamil Nadu from Punjab.

Fifteen apparently healthy Kandari cross-bred cattle aged about 4 years were purchased from Rurki, Patiala district of Punjab by a private dairy farmer in Erode, Tamil Nadu. Four animals showed eruptions on the lateral thoracic and dorsal abdominal regions of the body after 15-day period of quarantine. Manual palpation of the eruptions resulted in the emergence of larval stages of dipteran flies, identified by their morphology as Hypoderma from these animals. Molecular identification based on mitochondrial cytochrome oxidase-1 (COX-1) gene confirmed it to be Hypoderma lineatum. Treatment with oral ivermectin did not have any curative effects, with exacerbation of disease being noticed, as more than 500 eruptions subsequently emerged in each animal, which had to be culled. Consequences of long distance migration of host on parasite epidemiology are discussed. Awareness must be created among livestock farmers to prevent their economic loss while purchasing cattle from different parts of the country.

Genetic evaluation of growth in farmers' flocks of Madras Red sheep under long-term selection in a group breeding scheme.

The Network Project on Sheep Improvement (NWPSI)-Madras Red field unit is a group breeding scheme involving 198 farmers' flocks of Madras Red sheep in which selection for growth traits and rotation of rams have been practised for over two decades. Growth data collected from these flocks were used to evaluate the performance and understand the direct and expected responses to selection based on genetic parameters. The body weight at birth (BW), weaning weight (WW), 6-month weight (6W), 9-month weight (9W), 12-month weight (YW), pre-weaning average daily gain (ADG1, birth to 3 months), post-weaning ADG2 (3-6 months), ADG3 (6-9 months), ADG4 (9-12 months) and ADG5 (3-12 months) were 2.67, 10.05, 14.56, 18.36, 21.36, 80.13, 49.05, 43.00, 34.21 and 41.18 g, respectively. Univariate analyses were carried out using animal and sire models to estimate variance components. Heritability obtained from animal model for BW was 0.36 and the values for other body weight traits were almost unity. Heritability estimate for pre-weaning ADG1 was 0.31. Very high genetic variability was observed in spite of long-term selection and this sustenance of variability is one of the main advantages of a group breeding scheme, combining several flocks of smaller size. An increasing genetic and phenotypic trend was noticed for almost all the traits studied. The expected responses calculated based on genetic parameters also indicated scope for improvement.

Influence of sustained deworming pressure on the anthelmintic resistance status in strongyles of sheep under field conditions.

Anthelmintic resistance (AR) status in Madras Red sheep from selected field flocks of a government funded scheme, covered by regular, sustained anthelmintic treatment for more than 10 years was determined. Parameters such as fecal egg count reduction test (FECRT), larval paralysis assay (LPA), and allele-specific-PCR (AS-PCR) were used to test the efficacy of fenbendazole, tetramisole, and ivermectin at recommended doses, in two seasons. Sheep belonging to non-beneficiary farmers were used as controls. Mean FECRT values of beneficiary group during winter and summer seasons were 77.77 and 76.04, 93.65 and 92.12, and 95.37 and 98.06 %, respectively, for fenbendazole, tetramisole, and ivermectin. In the non-beneficiary groups, the corresponding values were 74.82 and 81.09 %, 96.05 and 97.40 %, and 97.26 and 98.23 %, respectively. The results revealed resistance to fenbendazole, suspect resistance to tetramisole and susceptibility to ivermectin in beneficiary flock. In non-beneficiary flock, while resistance was noticed against fenbendazole, both tetramisole and ivermectin were effective. FECR values were found to be significantly different between beneficiary and non-beneficiary groups against tetramisole. The results of LPA confirmed this finding, as 50 % of the Haemonchus contortus larvae were paralyzed at the concentration of 0.0156 µg/ml in the beneficiary group, while those of non-beneficiary groups required lower concentrations of 0.0078 µg/ml. AS-PCR revealed the predominance of heterozygous susceptible population of H. contortus in the beneficiary group. In this study, resistance to fenbendazole was confirmed in both the beneficiary and non-beneficiary groups and this could be attributed to frequent use of benzimidazoles as seen from the deworming records. Emergence of tetramisole resistance was detected in the beneficiary group, where the drug was used continuously for 4 years. Ivermectin was found to be effective in all the flocks. It is recommended that the practice of routine deworming of three to four times a year should be avoided, as it can lead to emergence of anthelmintic resistance.

Canine hepatic calodiosis with cirrhosis.

The nematode parasite Calodium hepaticum (Capillaria hepatica) has a global distribution and is commonly reported in rodents (definitive host), dogs, cats and wild animals. Humans especially children are more susceptible to the parasitic infection. This paper documents an incidental finding of hepatic calodiosis with cirrhosis in a stray dog and discusses the zoonotic implications. A non descript dog was brought for necropsy examination to the Department of Veterinary Pathology, Madras Veterinary College, Tamil Nadu, India. Liver appeared dark brown, mottled with multifocal random variably sized, grey white flat firm areas. Histopathologically, liver tissue revealed multiple random encysted large collection of eggs surrounded by mild inflammation with a few lymphocytes, macrophages and fine fibrosis. The eggs had characteristic barrel shape, bipolar ends, bilayered wall, cross striations between the walls, and yolk. Periodic acid Schiff stain demonstrated the glycolic wall of ova. Marked portal to portal fibrosis was demonstrated by Masson's trichrome (for collagen) and by Warthin-Starry (for reticulin) stains. The stage of parasitic infection was diagnosed as intermediate to chronic due to fibrosis. A need to study the prevalence of the disease in rodents, human and animals is emphasized. Improper burial of carcasses of rodents and dogs may contribute to spread of infection. Pets and stray animals may transmit infection to human and pose health risk.

Innovative way to dispense pheromones for off-host control of Rhipicephalus sanguineus sensu lato ticks.

Vapour patches dispensing pheromones were evaluated as lures to increase the attractiveness of sticky tick traps for Rhipicephalus sanguineus sensu lato (s.l.). Sex pheromone (SP), assembly pheromone (AP) and a combination of SP + AP at optimal concentrations were impregnated in vapour patches. The responses of the different stages of R. sanguineus s.l. (i.e. larvae, nymphs and adults) to the pheromones were evaluated using a Petri dish bioassay. The impregnated vapour patches were retained as such for a period of two mo and their efficacy was reassessed. In a subsequent field trial, pheromone impregnated vapour patches were placed as lures in bamboo (Bambusa vulgaris) sticky traps designed for the control of ticks in dog kennels. In vitro AP impregnated vapour patches were effective in attracting the different life stages of R. sanguineus s.l. whereas SP was effective in attracting the unfed and fed male stages of R. sanguineus s.l. The field trial revealed that questing and engorged larvae, nymphs and females of R. sanguineus s.l. were attracted more towards AP impregnated vapour patches than SP and AP + SP impregnated vapour patches. Fed and unfed male ticks were lured effectively by SP alone. The combination of SP + AP revealed no potent additive or synergistic effect.

Sheep pox disease outbreaks in Madras Red and Mechery breeds of indigenous sheep in Tamilnadu, India.

Sheep pox disease outbreaks were recorded among Madras Red (n=145) and Mechery (n=80) breeds of indigenous sheep on three farms in Tamilnadu. Over both breeds, adult mortality rate ranged from 2.66% to 37.5% and lamb mortality ranged from 10% to 17.33%. However, mortality was more in Mechery sheep (50% overall; 37.5% adults, 12.5% lambs) than in Madras Red sheep (24.28% overall; 10.34% adults, 13.79% lambs). The clinical signs observed were high fever, anorexia, respiratory distress, mucopurulent nasal discharge and in a few cases diarrhoea. Cutaneous lesions were mainly observed around nostrils, eyes, lips, ears and in the abdomen. Most of the lesions were covered with purulent materials and on cleaning with sterile swabs, fresh wounds were observed. Dry scabs were also observed over the oral commissure and maxillary areas, which on removal exposed fresh wounds. Important observations on necropsy were severe nodular lesions in the lungs and intestine. The disease was diagnosed as sheep pox by agar gel immunodiffusion test, isolation of virus and its neutralization in BHK(21) cells by specific antiserum and by electron microscopy.

Prevalence and staining characteristics of Blastocystis isolates from food animals in Tamil Nadu.

The present study was undertaken to investigate the prevalence and staining characteristics of Blastocystis isolated from food animals. Smears of the duodenal and caecal mucosal scrapings, collected from food animals, were stained with Giemsa, Gram's, modified acid-fast and acridine orange. Blastocystis was identified in 295 samples, including faeces and intestinal contents of animals like small ruminants (95), poultry (170) and pigs (30). The prevalence in pigs was found to be high (94.4%) followed by poultry (29.4%) and small ruminants (14%). Various forms of Blastocystis such as vacuolar, granular and amoeboid forms were identified by using different stains. The parasites stained with Giemsa were identified by the presence of eosinophilic nucleus and basophilic cytoplasm. In organisms stained with Gram's stain, the cytoplasm of the vacuolar forms took up the counter stain safranine. Blastocystis appeared as a pink colored cyst against bluish green background with modified acid-fast staining. The study shows that there is a very high prevalence of Blastocystis among the food animals investigated. Simple parasitological procedures, including direct microscopical examination and staining with agents like Giemsa, Gram's and acridine orange can assist identification of the parasites from intestinal contents and faecal material.

Seroprevalence of Neospora caninum antibodies in cattle and water buffaloes in India.

Neospora caninum is now recognized as a major cause of abortion in cattle worldwide, but there is no report of N. caninum infection in cattle in India. Serum samples from 427 dairy cattle and 32 dairy water buffaloes from 7 organized dairy farms located in Punjab, India, were tested for N. caninum antibodies using a commercial monoclonal antibody-based competitive enzyme-linked immunosorbent assay (ELISA). Antibodies to N. caninum were found in 35 of 427 cattle from 6 of the 7 farms; 9.6% of cows, 5.1% of heifers, and 5.0% of calves were seropositive, suggesting postnatal transmission of N. caninum on the farm. Antibodies to N. caninum were found in 16 of 32 buffaloes tested from 2 dairy farms. In total, 64 cattle and 16 buffalo sera already tested by ELISA were also evaluated by an indirect fluorescent antibody test (IFAT) to verify ELISA results. Of the 64 cattle samples, 29 sera were negative by both tests and of the 35 ELISA-positive sera, 12 had IFAT titers of 1:100 or higher (1 had IFAT titer of 100, 2 had IFAT titer of 200, and 9 had IFAT titers of 400 or higher). Of the 16 buffalo sera positive by ELISA, 1 had an IFAT titer of 1:400. Thus, antibodies to N. caninum were demonstrated in cattle sera by 2 serologic methods. To our knowledge this is the first report of N. caninum infection in cattle and buffaloes in India.

Investigations into the role of rats as intermediate hosts for Neospora caninum in Chennai, India.

Attempts were made to detect Neospora caninum in rats (Rattus rattus) in and around Chennai, India. During the study, 112 feral rats were trapped and blood, brain, heart, lungs and diaphragm samples were collected for serological, parasitological and molecular identification of N. caninum. Out of 112 rats, cyst-like structures were identified in 16 brain squash samples. However, the identity of the cysts could not be confirmed as N. caninum. A total of 12 sera samples were positive for N. caninum by indirect fluorescence antibody test (IFAT). Four of the 'cyst' positive samples were also positive by IFAT. None of the above samples showed amplification of N. caninum (Nc5) or toxoplasmatiid (ITS-1) fragments by PCR. In conclusion, the present study showed 10.71% seroprevalence of N. caninum among feral rats, which is a first report in India. Low prevalence of the organism in the environment and the consequent low chance of exposure of rats to N. caninum might explain the failure to detect the DNA in any of the samples tested in the study.

Isolation and characterization of Toxoplasma gondii from small ruminants (sheep and goats) in Chennai City, South India.

The present study aimed for the isolation and genotyping of Toxoplasma gondii from small ruminants (sheep and goats). 14 out of 193 tissue samples (either brain and heart) tested positive by MDAT for anti-T. gondii antibodies, were selected and bioassayed, which resulted 4 samples positive for T. gondii after 40 days of post inoculation. Four samples consisting of 3 numbers of sheep and 1 number of goat tissues out of 14 samples detected by B1 PCR, were genotyped at SAG3 locus by nested polymerase chain reaction-restriction fragment length polymorphism technique (nPCR-RFLP). The results of the present study revealed that the four isolates designated as TgShIn19, TgShIn76, TgShIn77 and TgGtIn27 were circulating in small ruminants, were belonged to genotypes of type II (TgShIn19) and type III (TgShIn76, TgShIn77 and TgGtIn27) which are in concordance with the previously reported genotypes from other animal species and further this presumptive results indicating that the genotype II and III could be the predominant in different animal species including birds and humans in India.

A novel assembly pheromone trap for tick control in dog kennels.

A novel ecofriendly sticky tick trap device for the control of dog tick Rhipicephalus sanguineus using gold nanoparticle assembly pheromone complex as a bait was developed. Assembly pheromones comprising of guanine, xanthine and adenine in the ratio of 25:1:1 was encapsulated in gold nanoparticle. The response of the different stages of unfed R. sanguineus ticks was evaluated using petridish bioassay. Statistical analysis was done using chi-square test. Petridish bioassay with unfed stages of R. sanguineus revealed that 100% of the larvae, nymph and adults were attracted to assembly pheromone nanogold complex within 24h. Of the 952 ticks trapped, ticks of different stages trapped in total by the baited sticky trap device, 543 (57%) were engorged and 409 (43%) were unfed ticks. The study revealed that assembly pheromone baited traps has the potential to control tick infestations in dog kennels.

Strobilocercus fasciolaris infection with hepatic sarcoma and gastroenteropathy in a Wistar colony.

Tapeworm cysts were identified in liver of Wistar rats and it induced fibrosarcoma in liver and gastroenteropathy in stomach and intestine. The tapeworm larva was confirmed as Strobilocercus fasciolaris by PCR linked mitochondrial DNA sequencing. Light microscopy, special staining (masson trichrome) and immunoflouresence supported the diagnosis of fibrosarcoma. Infiltration of plasma cells, macrophages and eosinophils were observed in the liver section. Gastric mucosal hyperplasia, dilation of gastric glands with secretion, intestinal mucosal cell hyperplasia, proliferation of duodenal submucosal glands were confirmed by light microscopy and supported by PAS, AB staining. The concomitant development of hepatic sarcoma and gastroenteropathy by larvae of Taenia taeniaeformis (S. fasciolaris) infection is very rare and is the first reported case in Wistar rats to our knowledge.

Detection of Toxoplasma gondii in small ruminants in Chennai using PCR and modified direct agglutination test.

A total of 193 sera samples, along with tissues (lung, heart, and brain) collected from 136 sheep and 57 goats from the Corporation slaughter house, Madras Veterinary College teaching hospital, and private mutton shops from Chennai were tested for Toxoplasma gondii. All the sera samples were tested using modified direct agglutination test. Of the 193 sera samples, 57 (29.5 %) had a minimum titre of 1:20, with 30.14 % (41/136) of sheep and 28.07 % (16/57) of goats being seropositive. Tissue samples from all 193 animals, when subjected to B1 based PCR to detect T. gondii DNA, showed 3.67 and 3.50 % to be positive in sheep and goats, respectively. In the present investigation B1 based PCR detected T. gondii in low numbers, possibly due to limitation of the sample size. The presence of T. gondii in tissues of sheep and goats slaughtered for human consumption in Chennai indicates the role of these food animals as potential sources of infection to human.

Emergence of Porcine Circovirus 2 Associated Reproductive Failure in Southern India.

Incidence of unusually high numbers of stillbirths was observed at a piggery unit at the Veterinary University research farm in Tamil Nadu State of India. Systematic examination of the tissue from stillborn piglets led to the identification of presence of Porcine circovirus 2 (PCV2). Detailed analysis utilizing electron microscopy, polymerase chain reaction and sequencing confirmed the presence of PCV2 in the tissue of affected piglets. Histopathology analysis of the affected piglet tissue showed lymphoid cell depletion of lymphnodes, spleen and infiltration of liver, kidney, myocardium, etc. Retrospective examination of the morbidity and mortality history in the farm revealed high mortality in young and weanling piglets suggestive of PCV2 infection-induced diseases. This is the first report of emergence of major disease incidence in farmed swine due to PCV2 infection in India.

Genotyping and detection of multiple infections of Toxoplasma gondii using Pyrosequencing.

A Pyrosequencing assay, based on SAG2 gene polymorphisms, was designed for genotyping and detection of multiple infections of Toxoplasma gondii. The assay was tested on samples spiked with DNA from single and multiple genotypes of T. gondii and also on a DNA sample from the brain of a rat with multiple infections. To evaluate the comparative efficacy of the assay, identical samples were also analysed by PCR-restriction fragment length polymorphism (RFLP) and dideoxy sequencing. The Pyrosequencing assay was found to be superior to the two conventional techniques. Genotyping and detection of multiple alleles were possible after a single PCR assay in duplex format, from both the spiked and direct samples. The simplex PCR assay enabled accurate quantification of the different alleles in the mix. In comparison, PCR-RFLP and dideoxy sequencing were neither able to unequivocally detect multiple genotype infections, nor quantify the relative concentrations of the alleles. We conclude that Pyrosequencing offers a simple, rapid and efficient means for diagnosis and genotyping of T. gondii, as well as detection and quantification of multiple genotype infections of T. gondii.

Redescription of Besnoitia bennetti (Protozoa: Apicomplexa) from the donkey (Equus asinus).

Besnoitia bennetti tissue cysts were found in four naturally-infected donkeys (Equus asinus) from the USA. Infectivity of its bradyzoites and tachyzoites to animals and cell culture was studied. The bradyzoites were not infectious to out-bred Swiss Webster mice, rabbits or gerbils. When fed tissue cysts, cats did not excrete oocysts. However, the parasite was infectious to interferon-gamma gene knock out mice. The parasite from tissues of two donkeys was grown successfully in bovine monocyte monolayers for the first time. Non-dividing, uninucleate tachyzoites were approximately 6 x 1.5 microm in size. Longitudinally-cut bradyzoites in tissue sections measured 8.7 x 1.9 microm. Ultrastructurally, tachyzoites and bradyzoites were similar to those in other Besnoitia species, and in particular to parasites described from cattle (Besnoitia besnoiti) and reindeer (Besnoitia tarandi), in that their bradyzoites lacked enigmatic bodies. Based on comparative analysis of three portions of nuclear ribosomal DNA (the small and large subunits and the first internal transcribed spacer) B. bennetti was found to be more closely related to the other congeners described from ungulates. The parasite was formally redescribed and specimens deposited in the US National Parasite Collections.

Differential detection of Hammondia hammondi from Toxoplasma gondii using polymerase chain reaction.

Hammondia hammondi and Toxoplasma gondii are two related coccidian parasites, with cats as definitive hosts and warm-blooded animals as intermediate hosts. It is difficult to differentiate them by morphological and serological parameters. In the present study, primers were designed to specifically amplify the ITS-1 region of H. hammondi to differentiate it from T. gondii. Attempts were made to detect the presence of H. hammondi DNA in the tissues of mice infected with H. hammondi alone, as well as from mixed infections with T. gondii, using the newly designed primers. The de novo primers effectively amplified the H. hammondi-specific target fragment from all samples containing H. hammondi, including those with concomitant T. gondii infection. Further, the primers did not amplify any fragment from the related parasites like T. gondii, Neospora caninum and Hammondia heydorni. The new primers provide simple and efficient means to differentially diagnose H. hammondi from T. gondii even in samples containing both parasites, thus obviating the need for other labourious techniques like mouse bioassay and in vitro cultivation.

Isolation of Neospora caninum from naturally infected white-tailed deer (Odocoileus virginianus).

Attempts were made to isolate Neospora caninum from naturally infected white-tailed deer (Odocoileus virginianus). A total of 110 deer killed during the 2003 hunting season in Virginia region were used for the isolation of N. caninum. Of these, brains from 28 deer that had NAT titer of 1:200 were inoculated into interferon-gamma gene knock out (KO) mice. N. caninum was isolated from the tissues of three deer and all three isolates were mildly virulent to KO mice. Only one of the isolates could be adapted to in vitro growth. Protozoa in the tissues of KO mice reacted with N. caninum-specific polyclonal antibodies and N. caninum DNA was demonstrated in infected tissues by PCR assays; sequences of portions of the ITS-1 and gene 5 loci were identical to those in the public database. This is the first record of in vitro isolation of N. caninum from white-tailed deer and lends credence to the white-tailed deer as an intermediate host for this parasite.