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Determination of Maternal Rabies Virus Neutralizing Antibody in Young Puppies.

Byline: Uzma Farid Durrani, Sagar Mal Goyal, Asim Khalid Mahmood, Cathleen Hanlon, Susan Moore, Muhammad Waqas and Raheela Akhtar


A pre-vaccination serological study was conducted on 100 puppies (0.5 IU/mL) in 29 puppies while 48 puppies showed mRVNA titer less than minimum protective level (0.2-0.4 IU/mL). RFFIT did not reveal mRVNA in 23 puppies that were declared lacking maternal rabies immunity. One basis of findings of this study it was concluded that puppies population lacking maternal rabies immunity is one of the responsible factors for increasing incidence of rabies in humans and dogs living in Lahore, Pakistan.

Key words

Maternal rabies virus neutralizing antibody (mRVNA), Rapid fluorescent focus inhibition technique (REFFIT), Puppies.

Pakistan is a rabies endemic country where 99% cases of rabies are followed by the rabid dog exposure every year (Durrani et al., 2012). These prevailing circumstances have urged the people for rabies prophylaxis of their pet dogs. Both active and maternal (passive) immunities are important for protection against rabies. In puppies, whelped by rabies vaccinated dams; maternal rabies virus neutralizing antibodies (mRVNA) are transferred to puppies. Maternal immunity (RVNA) of puppies persists till 3 months age (Morters et al., 2015; Hogenesch et al., 2004).

In Pakistan currently there is no test available to determine the mRVNA and in routine clinical practice primary vaccination of puppies is started at 3 months age to avoid interference by mRVNA (Durrani et al., 2012; Kasempimolporn et al., 1996). This serological ignorance of potential cases of maternal immunity failure is posing a serious risk of rabies spread to humans and other animals ultimately leading to the hike in rabies toll.

Rapid fluorescence focus inhibition technique (RFFIT) is a gold standard technique for the determination of RVNA titers (Bahloul et al., 2002). The test has good specificity (100%) and reproducibility (P>0.05) and is highly reliable in detecting rabies immunity status of dogs (Yu et al., 2012).

The present study was conducted to determine the mRVNA titers of young puppies at 1-3 months age and compare the effect of age on mRVNA titer.

Materials and methods

This study was conducted on 1-3 months old 100 healthy puppies of either sex. Puppies belonged to different areas of Lahore, Pakistan that is a rabies endemic city. Selection of puppies was made on the basis of rabies vaccination history of their dams and consent of the owners. Data about age, health status and medication history of puppies was collected for each puppy. Puppies were divided into 3 age based groups i.e. groups of 1 month, 2 months and 3 months old puppies.

Blood samples were collected from cephalic vein. Sera were separated, stored at -80degC before shipping these to K-State Rabies Laboratory at Kansas State University, Manhattan, K.S., USA for mRVNA screening by rapid fluorescent focus inhibition technique (RFFIT). For this analysis 11 ul of serum samples were placed in a water bath at 56degC for 1 h to inactivate complement (Kramer et al., 2009). Three serial fivefold dilutions of test Sera and a 2 IU/mL stock solution of standard serum prepared in RFFIT media were transferred to 8-well Lab Tek chamber slides followed by addition of 100uL of pre-diluted CVS-11 (CCID50). The mixture was incubated at 37degC for 90 min. followed by the addition of 200 uL of baby hamster kidney cells suspension (5x105 /mL). After 24 h of incubation at 37degC, the chamber slides were fixed in 80% cold acetone followed by a rinse in phosphate buffer saline (PBS) for 3 min.

After drying in a bio safety cabinet for 10 min, 156 uL of fluorescein conjugated rabies antibody (Kramer et al., 2009) was added to all wells and incubated at 37degC for 45 min. followed by PBS washing and then dried. Under a fluorescent microscope, 20 microscopic fields were examined in each well of the slide at 200X magnification. Number of fields containing at least one fluorescent cell was counted starting at the same corner of the slide and taking four rows of the five fields. Control slides were read first and the number of positive fields in each serum dilution was recorded.

Table I.- Age based RFFIT findings reveal protective mRVNA titers in 14, 11 and 4 puppies that were 1, 2 and 3 months old, respectively while mRVNA titer was below protective level in 20, 16 and 12 puppies at 1, 2 and 3 months age, respectively. Immunity failure was observed in 13, 5 and 5 puppies those were 1, 2 and 3 months old, respectively.

Age (months)###mRVNA titer (IU/mL)

###[greater than or equal to] 0.5###0.2 - 0.3###[?] 0.2





There were two serum samples per slide and end point titer of the test serum samples was calculated on the basis of number of positive fields in corresponding dilution (1:5) using Reed and Muench calculation chart (Reed and Muench, 1938), according to which dilution field with viruses 1:5, 0/20, 1/20 and > 2/20 has titer of 0.1, 0.1 and < 0.1, respectively.

mRVNA concentration (IU/mL) for test serum samples was calculated by using the following formula:

End - point titer of test serum/point titer of reference serum x 2.0 IU/mL of reference serum

Data were statistically analyzed using Student's t-test and One Way Analysis of Variance (ANOVA) and multiple comparisons were carried out by using Least Significant Difference test (Snedecor and Cohran, 1967). Confidence level was 95% and the P value was considered significant at < 0.05.

Results and discussion

The screening of mRVNA titer is an effective tool to determine the rabies protective immunity in animals and humans. RFFIT is a WHO recommended gold standard serological assay used for RVNA determination (Durrani et al., 2012).

The under discussion study was conducted on 100 puppies (1-3 months old) whelped by regularly rabies vaccinated dams. RFFIT revealed a variation of mRVNA titers among different age groups of puppies (Table I). Statistical analysis proved a significant difference (P0.5 IU/mL till 36 days after birth and then this level starts declining to <0.5 IU/mL. On the basis of these observations they suggested that in rabies endemic areas all dogs, including those less than 3 months age, must be vaccinated with high quality inactivated rabies vaccine to secure the animal and human population against rabies. Puppies from non-vaccinated dams respond well to vaccination after the 4-10 weeks of age with a progressive increase in RVNA titer. On the contrary, puppies from vaccinated dams respond only at 10 weeks of age, although detectable mRVNA titer decreases by 6 weeks (Moore and Hanlon, 2010; Rashid et al., 2009; Winters, 1981). It is also reported earlier that a continuous use of corticosteroids, immune-suppressive agents and some health disorders can interfere the immunity level (Rashid et al., 2009).


It is concluded that there is a high risk of rabies maternal immunity failure under field conditions and a significant relation exists between mRVNA titers and age of the young puppies. In this scenario it is recommended that for puppies under 3 months age sero-screening must be practiced to confirm the mRVNA titer prior to especially in rabies endemic areas. RFFIT is a gold standard diagnostic assay for mRVNA determination however, currently this diagnostic technique is not available in Pakistan and here rabies antibody titer is diagnosed on the basis of ELISA.


We are highly grateful to Prof. Dr. Gary Anderson, Director Veterinary Diagnostic Laboratory, Kansas State University, USA for accommodating this project at their laboratories as well as for their cooperation during this study. We also acknowledge the financial assistance by Higher Education Commission of Pakistan under International Research Support Initiative Program to perform RFFIT in the U.S.A.

Statement of conflict of interest

Authors have declared no conflict of interest.


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Article Details
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Author:Durrani, Uzma Farid; Goyal, Sagar Mal; Mahmood, Asim Khalid; Hanlon, Cathleen; Moore, Susan; Waqas,
Publication:Pakistan Journal of Zoology
Article Type:Report
Geographic Code:9PAKI
Date:Jun 30, 2017
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