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Research in Veterinary Science 84 (2008) 250–253

Short Communication

Serological evidence of Neospora caninum infection in mithun(Bos frontalis) from India

S. Rajkhowa *, C. Rajkhowa, P.R. Dutta, Paihem Michui, R. Das

National Research Centre on Mithun, Indian Council of Agricultural Research, Jharnapani, Nagaland 797 106, India

Accepted 7 May 2007

Abstract

Studies conducted on mithuns maintained at National Research Centre on mithun, Indian Council of Agricultural Research (ICAR),Nagaland, India and mithuns found in free-ranging condition of Nagaland revealed that the overall prevalence of antibodies to Neospora

caninum in mithun was 10% (95% CI = 5–15) when detected by a commercially available competitive enzyme-linked immunosorbentassay test. Highest (prevalence rate = 16, 95% CI = 8–24) seroprevalence was found in mithuns above 3 years of age and lowest (prev-alence rate = 2, 95% CI = 0–6) in mithuns of 2–12 months old. No statistically significant difference was observed between male (prev-alence rate = 7, 95% CI = 0–14) and female (prevalence rate = 12, 95% CI = 6–18) seroprevalences. The seroprevalence was found tobe higher (prevalence rate = 20, 95% CI = 9–31) in mithuns found in free-ranging condition in comparison to mithuns kept in semi-intensive system (prevalence rate = 5, 95% CI = 1–9). This is probably the first report on serological evidence of N. caninum infectionin mithun. The possible role of sylvatic fauna in the epidemiology of N. caninum infection mithun is also discussed in brief.� 2007 Elsevier Ltd. All rights reserved.

Keywords: Bos frontalis; Mithun; Neospora caninum; Risk factors; Seroprevalence

The mithun (Bos frontalis) is a unique ruminant found inthe hill regions of northeast India, Myanmar, Bhutan, Ban-gladesh, China and Malaysia. The Indian gaur (Bos gau-

rus); also known as the ‘‘Indian bison’’ and as the‘‘gayal’’ is the wild ancestor of mithun. Chromosomally,the gaur and the mithun are identical (Gupta et al.,1999). This animal is well adapted in steep jungles at an ele-vation ranged from 300 to 3000 m above sea level (m.a.s.l.)and has got important place in economic, social, culturaland religious life of tribal people inhabiting the region.This is an underutilized animal and has great potentialfor quality meat, milk and leather production. This animalis basically used for beef purpose. Mithun produces milkwith fat percentage ranging from 11 to 13% (AnnualReport, 1999–2000) and the milk-production potential ofthis animal was reviewed (Nath and Verma, 2000; Bujar-

0034-5288/$ - see front matter � 2007 Elsevier Ltd. All rights reserved.

doi:10.1016/j.rvsc.2007.05.002

* Corresponding author.E-mail address: s_rajkhowa@lycos.com (S. Rajkhowa).

baruah et al., 2002). The mithun prefers moderate climate,dense forest and steep slopes, which are the general geo-graphical features of this region. This animal is still keptin the forest under a free-grazing system, where there isnobody who can look after them. Neospora caninum

(N. caninum) is an obligate intracellular protozoan thatcan infect domestic and wild canids, as well as ruminantsand equines (Oliveira et al., 2004). There are meager reportson N. caninum infection in other species of animals in Indiabut none from mithuns. This is probably the first report onseroprevalence of N. caninum antibodies in mithuns. Themain objectives of the study were: (i) to determine the sero-prevalence of N. caninum antibodies in serum of mithunsfrom India and (ii) to analyze the association between sero-prevalence and age, sex and system of management.

This study was conducted on mithun farms of theNational Research Centre on Mithun, in Nagaland inIndia (where mithuns from four mithun rearing states arekept) and mithuns that are found in their natural habitat(free-ranging system) in the state of Nagaland. The state

Table 1Seroprevalence of antibodies to N. caninum in mithun in India accordingto age, gender and system of management

Factor n Prevalence(%)

95%CI

OR 95%CI

P-value

Age

2–12 months 42 2 0–6 1.00 0.011–3 year 38 5 0–12 2.27 0–26>3 year 79 16 8–24 8.07 1–64

Gender

Male 55 7 0–14 1.00 0.39Female 104 12 6–18 1.66 1–5

Management system

Semi-intensive 105 5 1–9 1.00 0.004Free-ranging 54 20 9–31 5.11 2–16

Total 159 10 5–15

Results of univariate analysis presented as odds ratio (OR) and 95%confidence intervals (CI).

S. Rajkhowa et al. / Research in Veterinary Science 84 (2008) 250–253 251

Nagaland is located in the extreme North-east of the coun-try and lies between latitude 25�10 0 and 27�4 0N and longi-tude 93 �15 0 and 95�15 0E. The altitude varies from 194 to3826 m.a.s.l. Average annual rainfall in the state is2000 mm. Summer mean temperature varies from 15 to30 �C and winter temperature ranges from 4 to 25 �C.

One hundred and fifty nine (male = 55; female = 104)mithuns (semi-intensive = 105; free-ranging = 54) wereused for the study. The animals of the farms were keptunder semi-intensive system of management and allowedto graze in the forest areas of the farm specially developedfor grazing of mithun. During the night the mithun weretethered in their respective sheds. In addition to sufficientgreen fodder, their daily food rations normally included aconcentrate mixture, according to the body weight of theanimal. Though free-range mithuns are found in deep for-ests, they are periodically (at one month interval) broughtto the open field by their owners to offer salt to theirmithuns, the common practice of offering salt to themithuns by the mithuns rearers. Each individual mithunrearer has all relevant information about their mithun.The food rations of the free-range mithuns are mainly con-sisted of forest leaves, young plants and grasses. The seracollected from these animals were separated, numberedand stored at �20 �C until testing.

Sera were analyzed for antibodies to N. caninum by acommercially available competitive enzyme linked immu-nosorbent assay (cELISA) test (VMRD, Inc, Pullman,USA) using the manufacturer’s recommended procedure.The sensitivity and specificity of cELISA under field condi-tion were 96% and 99%, respectively. In brief the test wascarried out as follows: Fifty micro liter of undiluted serumsample was added to each well of the 96-well antibodycoated micro titration plate and positive and negative con-trols in duplicate were also included in the test. The nega-tive and positive controls used in the test were of bovineorigin. The plate was then incubated uncovered at roomtemperature for 1 h. After 1 h of incubation the plate waswashed three times with 1· wash solution. Fifty micro literof diluted antibody-peroxidase conjugate (the conjugate isa monoclonal antibody directed against N. caninum andconjugated to HRPO) was added to each well and the platewas incubated uncovered at room temperature for 20 min.After the 20 min incubation, the plate was again washedthree times with 1· wash solution. Fifty micro liter of sub-strate solution (TM blue) was added to each well and thenthe plate was incubated uncovered at room temperature for20 min. Fifty micro liter of stop solution (sodium fluoride)was added to each well followed by gentle mixing of thewell contents. Immediately after addition of stop solutionthe reading was taken with the help of an ELISA readerusing 620 nm filter. The percentage inhibition (%I) for eachtested sample was calculated by applying the followingformula:

%I ¼ 100� ½ðSample O:D:Þ� 100=ðMean negative control O:D:Þ�

The following two criteria were used to validate the test:

(i) The mean of the negative controls must produce anoptical density P0.30 and <2.50.

(ii) The mean of the positive controls must produceP30% inhibition. The sample producing %I ofP30% was considered as positive.

Data were analyzed by using standard method (Snede-cor and Cochran, 1994). Chi-square (v2) test was used toanalyze the influence of all examined factors as indepen-dent categorical variables on N. caninum seroprevalence.The variables that were found significant at 5% (P < 0.05)level of significance in univariate analysis were onlyincluded in the multivariate logistic regression model.Results are presented as adjusted odds ratios (OR) with95% confidence intervals (CI). Analyses were done withSPSS 13.0 software for Windows with a probability (P)value <0.05 as statistically significant. Confidence limitsfor the proportions were established by exact binomial test.

Age, gender and farm management characteristics ofexamined mithun population are presented in Table 1.The overall seroprevalence of antibodies to N. caninum inmithun was found to be 10% (95% CI = 5–15). Univariateanalysis of risk factors showed that age and type of man-agement system are associated with N. caninum seropreva-lence in mithun. No statistically significant (P = 0.39)difference was observed between male and female seropre-valences. The seroprevalence increased with the increase inage of the animals. Highest seroprevalence was observed inmithuns above 3 years of age (prevalence rate = 16, 95%CI = 8–24) with the lowest seroprevalence recorded inmithun of 2–12 months of age (prevalence rate = 2, 95%CI = 0–6). Mithuns from free-ranging system (prevalencerate = 20, 95% CI = 9–31) were more likely to be seropos-itive than mithuns kept in semi-intensive system of man-agement (prevalence rate = 5, 95% CI = 1–9). These

252 S. Rajkhowa et al. / Research in Veterinary Science 84 (2008) 250–253

factors (age and type of management system) were simulta-neously analyzed in a logistic regression model to determinetheir relative contributions to N. caninum seropositivity,while adjusting for their effects (Table 2). Compared withthe respective reference categories, the likelihood of N. cani-

num infection was found to be almost eight times more inmithuns aged above 3 years of age (P = 0.05). Likewisemithuns from free-ranging system (P = 0.003) was foundto be almost six times more vulnerable to infection incomparison to mithuns kept in semi-intensive system ofmanagement.

The serological results of the study indicate thatmithuns in India are exposed to N. caninum infection.Although these findings could not be compared withany others, due to lack of other studies on N. caninum

infection in mithun, it was possible to compare them withfindings on other bovine species such as cattle and buffa-loes. The present study revealed that the mithuns of allage groups are susceptible to N. caninum infection. Anincrease in seroprevalence was evident in the oldermithuns. The present findings are in agreement with thefindings of Ragozo et al. (2003) and Guimaraes-Junioret al. (2004), possibly owing to the increased risk of infec-tion by horizontal transmission. This fact suggests theexistence of sporulated N. caninum oocysts in the environ-ment, which characterizes horizontal transmission, asobserved by Dijkstra et al. (2002). Guarino et al. (2000),in Italy, also found that N. caninum prevalence increasedwith the age of buffaloes, thus indicating exposure to theparasitic oocysts. This result may be explained by theincrease of horizontal postnatal oocyst infection withage described by Wouda et al. (1999) and Rinaldi et al.(2005) in cattle. Though various authors (Corbelliniet al., 2001; Wisniewski et al., 2002) had claimed that ver-tical transmission is the most common method of trans-mission for N. caninum infection, the present studysupports the horizontal transmission of this parasite,which could be due to inclusion of less number of animalsin different age groups of animals. Sex, however, had noinfluence on the seroprevalence of antibodies to N. cani-

num in mithuns. This indicates that under the same cir-cumstances, both male and female mithuns have an

Table 2Risk factors for N. caninum infection in mithun in India

Factor Adjusted OR 95% CI SE P-value

Age

2–12 months 1.001–3 year 1.64 0–21 1.25 0.70>3 year 7.92 1–65 0.53 0.05

Management system

Semi-intensive 1.00Free-ranging 5.96 2–19 0.56 0.003

Final logistic regression model. Results presented as adjusted odds ratio(OR) and 95% confidence intervals (CI).SE: Standard error.

equal chance of acquiring infection with N. caninum.From the present investigation, it was also observed thatmithuns found in free-ranging condition were more likelyto be seropositive than mithuns kept in semi-intensive sys-tem. The highest seroprevalence of N. caninum in free-ranging system could be due to existence of a sylvaticcycle of this parasite. Though various workers (Ferroglioet al., 2003; Rosypal and Lindsay, 2005) supported theexistence of a sylvatic cycle for this parasite, further stud-ies are needed to evaluate the existence of a sylvatic cycleof N. caninum. As intensive mithun farming is graduallypicking up by the mithun rearers in north-eastern hillregion of the country, the status of this animal in relationto different diseases will have strong bearing in formulat-ing strategies for disease investigation programme of largeruminants because of increase in chances of interminglingof this species with other ruminants. As sporadic abortionhas been recorded in mithun, further studies are requiredto know the role of N. caninum (if any) in causing abor-tion in mithun.

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