L. D. Singla

Rodents as reservoirs of parasites in India.

We monitored the prevalence of endoparasitic infections of rodents in Punjab State, India, between January 2004 and December 2005. Three species of wild rodents, namely the house rat, Rattus rattus (n= 42), the lesser bandicoot rat, Bandicota bengalensis (n= 34) and the Indian gerbil, Tatera indica (n= 15), were live-captured from houses and crop fields. Examination of various organs revealed that the highest rates of endoparasitic infection occurred in R. rattus (40.5%), followed by B. bengalensis (35.3 %) and then T. indica (20.0%), with an overall infection rate of 35.2%. All three rodent species were found naturally infected with one or more species of helminths. Metacestodes (1-6) of Cysticercus fasciolaris (larvae of Taenia taeniaeformis) were found in all three rodent species (in the liver). In one male T. indica, numerous robust T. taeniaeformis metacestodes were found in oval sacs attached to the mesentery and the abdominal wall, an unusual site. The cauda epididymal fluid of the same gerbil was also found to be infected with a very rare species of strongylid nematode, which could not be identified to genus or species level. It is possible that this nematode is transmitted sexually and thus may affect the reproductive potential of gerbils. This appears to be the first report of this phenomenon. In one B. bengalensis individual, the intestine was found to be obstructed with an acanthocephalan, Moniliformis moniliformis, with concurrent infection with C. fasciolaris in the form of multiple cysts in the liver. Although no natural protozoan infection was found in these field rodents, experimental Trypanosoma evansi infection has been established in all three species with high pathogenicity, and the possibility of sexual transmission was supported by the presence of T. evansi in the cauda epididymal fluid of male rats.

Vaccines against Toxoplasma gondii: Status, challenges and future directions

Toxoplasma gondii is a ubiquitous protozoan parasite that can infect a wide range of animals including humans. This single known species in the genus Toxoplasma is considered as one of the most successful eukaryotic pathogens which is of major medical and veterinary importance. Effective vaccines may contribute toward preventing and controlling the spread of toxoplasmosis. The present communication addresses the current status of development of vaccines against T. gondii. Further discussion is made on the difficulties along with challenges, such as vaccine construct, mode of vaccine administration and standardization of immunization evaluation. Finally suggestions are made on possible directions for future research on the development of vaccines against T. gondii.

Prevalence and haemato-biochemical profile of Anaplasma marginale infection in dairy animals of Punjab (India).

OBJECTIVE To do the systematic comparison of prevalence of anaplasmosis by PCR and Giemsa stained thin blood smear (GSTBS) based parasitological assays in dairy cattle of Punjab, which has not been reported yet. To analyse the haematobiochemical alterations in infected animals to arrive at the conclusion regarding the pathogenicity induced by Anaplasma marginale (A. marginale) in latent and patent infection. METHODS Study was conducted on 320 animals (236 cows, 62 calves and 22 buffaloes) of Punjab, India. PCR on genome of A. marginale was performed by targeting msp1 β gene using specific primers BAP-2/AL34S, amplifies products of size 407 bp. Questionnaires based data on the characteristics of the infected animals and management strategies of the farm were collected and correlated. RESULTS Higher prevalence and more significant association was observed in the PCR based molecular diagnosis (P=0.00012) as compared to that in GSTBS (P=0.028 8) based diagnosis with various regions under study. With respect to the regions, highest prevalence was recorded in Ferozepur by PCR based diagnosis, while that in Jalandhar by GSTBS examination. Similar marked significant association of the PCR based diagnosis with the age of the animals under study (P=0.00013) was observed elucidating no inverse age resistance to A. marginale in cow calves. Haematobiochemical profile of infected animals revealed marked anemia, liver dysfunction and increase globulin concentrate indicating rise in immunoglobulin level to counteract infection. CONCLUSIONS PCR is far more sensitive in detecting the disease even in latent infection which may act as nidus for spread of anaplasmosis to susceptible animals in endemic areas. Severity of anaemia and liver dysfunction were comparable both in patent as well as latent infection indicating pathogenicity of both.

Transplacental transmission of Babesia equi (Theileria equi) from carrier mares to foals

This article communicates the first per-acute and fatal clinical report of transplacental transmission of Theileria equi from carrier mothers to foals from Punjab, India. Two cases of equine piroplasmosis due to T. equi were diagnosed in neonatal foals borne to Throughbred mares. High degree of parasitemia with annular and maltese cross shaped parasites in erythrocytes was seen in both cases. Blood cellular changes revealed leucopenia and neutropenia with mild degenerative left shift. Anaemia was macrocytic normochromic type. Intense yellow discoloration of mucous membranes indicated jaundice. In areas endemic for equine piroplasmosis, jaundice in neonatal foal can be easily misdiagnosed as neonatal isoerythrolysis. Foals with post-partum jaundice should always be screened for equine piroplasmosis.

Molecular Prevalence of Babesia bigemina and Trypanosoma evansi in Dairy Animals from Punjab, India, by Duplex PCR: A Step Forward to the Detection and Management of Concurrent Latent Infections

Specific duplex polymerase chain reaction (PCR) was employed on 411 (386 cattle and 25 buffaloes) blood samples of dairy animals from 9 districts of Punjab, India, for simultaneous detection of Babesia bigemina and Trypanosoma evansi. The results were compared and correlated with conventional Giemsa stained thin blood smear (GSTBS) examination and haematological alterations to know the clinical status and pathogenicity of infections. The Bg3/Bg4 and TR3/TR4 primers were used in duplex PCR for B. bigemina and T. evansi amplified products of 689 bp and 257 bp, respectively. The overall prevalence by duplex PCR was found to be 36.49, 2.43, and 3.41% for T. evansi, B. bigemina, and dual infection, respectively. A more significant difference was observed for dual infection status (P ≤ 0.005) as compared to T. evansi (P ≤ 0.05) and B. bigemina (P ≤ 0.01) among various districts under study. A very low prevalence of T. evansi (0.73%) and B. bigemina (0.48%) was seen by GSTBS. The highly sensitive, specific, and cost-effective duplex PCR was able to detect latent T. evansi and B. bigemina infection in cattle and buffaloes. Haematological evaluation revealed marked pathology in B. bigemina infected group and in dual infected group in contrast to that infected with T. evansi alone.

Differential proteomic profiles from distinct Toxoplasma gondii strains revealed by 2D-difference gel electrophoresis

Toxoplasma gondii is an obligate intracellular protozoan that infects mammals and birds. Human infection during pregnancy may cause severe damage to the fetus. Reactivation of latent infection in immunocompromised patients can cause life-threatening encephalitis. T. gondii strains are highly diverse but only a few lineages (Type I, II and III) are widely spread. In mouse model, Type I strains are highly virulent, whereas Type II and III strains are intermediately or non virulent. It is not clear how much quantitative difference exists in proteomic profiles among these distinct T. gondii lineages. In the present study, the proteomic profiles of T. gondii tachyzoites from these lineages were investigated by two dimensional fluorescence difference gel electrophoresis (2D-DIGE) and mass spectrometry (MS) technologies. A total of 2321 protein spots were detected. Overall, the GT1 strain of Type I lineage and the strain PTG of Type II lineage have highly similar proteomic profiles and both are different from that of the CTG strain of Type III lineage. Eighty-four protein spots were differentially expressed by greater than 1.5-fold in relative abundance and 10 of them were identified to 7 T. gondii proteins in existing database. Investigation of the quantitative differences in proteomics among distinct T. gondii strains should facilitate our understanding of difference in biological processes and pathogenesis of distinct T. gondii genotypes, which will provide basic information to determine treatment regimen for different manifestation of toxoplasmosis.

Multiplex PCR for detection of Trypanosoma evansi and Theileria equi in equids of Punjab, India

Multiplex PCR for simultaneous detection of Trypanosoma evansi and Theileria equi in single-step reaction was optimized and employed on 108 equids (99 horses and 9 donkeys/mules) blood samples collected from two agro-climatic zones (Sub-mountain undulating zone and Undulating plain zone) of Punjab to evaluate the status of concurrent infection and associated risk factors. The amplification products of 257 and 709 bp targeting repetitive nucleotide sequence of variable surface glycoproteins of T. evansi and 18S rRNA gene of T. equi, respectively expressed high fidelity of the primer pairs with sequence homology to neighboring geographic isolates. The overall prevalence of T. evansi and T. equi was 3.7 and 1.85%, with Undulating plain zone at higher infection risk for T. equi (OR=3.24, 95% CI=0.28-83.65); and Sub-mountain undulating zone (OR=∞, 95% CI=0.25-∞) for T. evansi. Multiplex PCR revealed higher risk of infection of both T. equi (OR=6.75, 95% CI=0.58-175.38) and T. evansi (OR=2.11, 95% CI=0.05-80.36) in the farms with inappropriate management system. The risk factor associated with the type of host species had an odds ratio of 12.35 (95% CI=0.29-508.37) for donkeys/mules versus horses for T. evansi infection. This group was also at higher risk of infection with Odds ratio (OR) of 4 (95% CI=0.14-53.99) for T. equi. The current investigation brings out various commodities at risk of infection pertaining to equid trypanosomosis and theileriosis evaluated by a rapid and sensitive multiplex PCR assay.

Spatial distribution, risk factors and haemato-biochemical alterations associated with Theileria equi infected equids of Punjab (India) diagnosed by indirect ELISA and nested PCR.

Equine piroplasmosis is a febrile, tick-borne disease of equids predominately caused by obligatory intra-erythrocytic protozoa Theileria equi in the Indian sub-continent. A cross-sectional study was carried out on 464 equids (426 horses and 38 donkeys/mules) in Punjab, India to assess the level of exposure to equine piroplasmosis by 18S rRNA gene nested polymerase chain reaction (nPCR) and equine merozoite antigen-2 (EMA2) indirect-ELISA (enzyme linked immunosorbent assay), to investigate risk factors and haemato-biochemical alterations associated with the infection. The endemicity of the disease was confirmed by positive PCR amplification in 21.77% and positive antibody titers in 49.78% equid samples. There was a fair agreement between these two diagnostic techniques (Kappa coefficient=0.326). The spatial distribution analysis revealed an increasing trend of T. equi prevalence from north-eastern to south-western region of Punjab by both the techniques correspondingly, which proffered a direct relation with temperature and inverse with humidity variables. The relatively prominent risk factor associated with sero-positivity was the presence of other domestic animals in the herd, while the propensity of finding a positive PCR amplification was higher in donkeys/mules, animal kept at unorganised farm or those used for commercial purposes as compared to their counterparts. There was a significant increase in globulins, gamma glutamyl-transferase, total bilirubin, direct bilirubin, indirect bilirubin, glucose levels and decrease in total erythrocyte count, haemoglobin, packed cell volume by animals, which were revealed positive by nPCR (may or may not positive by indirect-ELISA) and increase in creatinine, total bilirubin, direct bilirubin, glucose and decrease in total erythrocytes count by animals, which were revealed positive by indirect-ELISA (alone). To our knowledge, this study, for the first time, brings out a comprehensive report on the status on spatial distribution of T. equi in Punjab (India) state, thoroughly investigated by molecular and serological techniques, evaluating various environmental and demographic risk factors along with the haemato-biochemical alterations in the exposed animals.

Retrospective study of clinical and hematological aspects associated with dogs naturally infected by Hepatozoon canis in Ludhiana, Punjab, India

OBJECTIVE To evaluate clinical and hematological aspects of dogs naturally infected with Hepatozoon canis (H. canis) presented at the Small Animal Clinics of Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana. METHODS Blood films of 34 naturally infected dogs were examined for haematological alterations and parasitaemia. Signalment and clinical signs were recorded from the animals. Clinical histories were filled out during the consultation. RESULTS Of the 34 positive dogs by Giemsa stained peripheral blood films, 88.23% presented parasitaemia by H. canis only, while 11.77% had the combination of H. canis, Babesia sp. and Ehrlichia sp. Young male dogs less than one-year-old, of non-descript breed, were the most commonly affected. And 26.47% were presented with anorexia/inappetence as the only clinical symptom. Other clinical symptoms were mild to moderate fever, pale mucosae and lethargy; a few were also showing the signs of vomiting and diarrhoea. Haematological alterations showed mainly normochromic-normocytic anaemia, leukocytosis and neutrophilia. CONCLUSIONS The findings of this study substantiate that H. canis caused clinical and haematological alterations of the varied intensity in dogs, even with low parasitaemia, should be taken into consideration.

Cytopathology of parasitic dermatitis in dogs

Out of 44 cases of dermatitis in dogs, 11 cases of parasitic origin were analyzed by cytopathology. Histopathologic examination of punch biopsies was also done for correlation with cytologic findings. Sarcoptic dermatitis was recorded in six cases, wherein, besides sarcoptic mites, neutrophils, macrophages, and plasma cells and keratinizing epithelial cells were also seen. Hematology revealed a relative neutrophilia and mild eosinophilia. Four cases of severe and generalized demodicosis complicated with bacteria and/or Malassezia sp. infection were also recorded. Histopathologically numerous Demodex sp. mites in varying stage of maturation were found damaging the hair follicles along with associated pathological changes and foreign body granulomas in one case. In addition, flea allergy dermatitis was also observed in one dog. In nutshell, cytology was found to be unequivocally effective in diagnosing parasitic dermatitis.