Evaluation of the shedding routes and serological patterns in experimentally-induced Brucella melitensis infection in dexamethasone-treated and transport-stressed goats

Aim: To identify and evaluate the shedding routes and patterns following experimentally-induced Brucella melitensis infection in dexamethasone-treated and transport-stressed goats. Materials and Methods: Twenty four healthy, adult goats were divided into 4 groups: A, B, C and D respectively. Group A 7 was treated with dexamethasone for 8 days prior to inoculation with 10 Colony Forming Units of B. melitensis via the intraocular route. Group B was transported for 3 hours prior to inoculation with a similar dose. Group C was inoculated with a similar dose without subjecting the animals to any prior treatment, and this group served as our positive control. Group D was not inoculated with the infective dose and served as our negative control. Blood samples along with nasal, ocular, and vaginal swabs were collected on days 0, 3, 7, 10, 14, and weekly thereafter until day 63 post inoculation (pi) and were analyzed by PCR, Rose Bengal Plate Test (RBPT), and indirect ELISA techniques. Results: The nasal, ocular and vaginal swabs tested positive for Brucellosis with PCR from day 7, with nasal route being the first and most consistent route to reveal the positive results. Group B showed the earliest onset of shedding the bacterium (day 7) followed by group A which started from day 10 and shed relatively more positive of the bacterium via the routes examined. Blood samples tested positive with PCR from day 7 through 14 and the results were inconsistent subsequently. Sera samples tested positive with RBPT on day 14 in all the 3 infected groups but more consistent in group C. On the other hand, tests using ELISA showed positive results from day 7 pi, with group C having a 100% seroconvertion –while groups A and B showed only 50% seroconvertion. Conclusion: The consistent shedding via the nasal, ocular, and vaginal routes in groups A and B implied possible immunosuppression in the infected animals. We recommend that programs designed to control Brucellosis should consider analyzing a larger number of biological samples to enhance the accuracy of identification of shedders.


Introduction
of the developing nations that have no effective vaccination-based control and slaughter programmes, Brucellosis is now recognized as one of the most such as the countries in Mediterranean, Middle East, important among the common zoonotic diseases across Africa, Latin America, and parts of Asia [7]. the world and is a disease that poses a major threat to Goats in the tropics are usually reared under the both human health and animal populations [1].The traditional extensive management system and are often World Health Organization considers it as a neglected transported over a considerable distance for marketing zoonosis, because adequate control programmes were and slaughter purposes [8,9].This long range transpornot planned in numerous countries despite its huge tation, an age old inevitable husbandry practice [10], impact not only on human and animal health, but also creates undue stress on the animals.In this regard, it is on the economy [2].Brucellosis, especially affecting important to note that the artificially-induced stress the food animals, poses a barrier to the trade of animals upon administration of dexamethasone has been and animal products between countries.This is because reported to mimic the stress elicited by natural causes Brucellosis causes considerable economic losses due [11,12,13].Studies investigating the effect of stress on to abortions and fertility problems, encountered often the productivity of small ruminants' have primarily in the sheep and goat industry [3,4,5].Brucellosis in focused on caprine pneumonia [14,15] with very little small ruminants, especially goats is caused by B.
emphasis on the elucidating the effect of stress on the melitensis [6] and the incidences of transmission to the transmission of zoonotic diseases.human population have significantly increased in most With a myriad of stress factors that goats are constantly subjected to, it is imperative to understand the interactions between stress, reproductive zoonotic disease occurrence, and transmission of the disease.
Understanding the interaction between the aforemen-for less than 15 minutes under a shade (for resting tioned factors is critical to enhance our knowledge base purposes).The goats were neither fed nor watered for proper planning and swift control of these diseases during the resting period.The animals were then in goats, especially in those herds that are constantly infected, after being off loaded, with doses that were subjected to environmental and managemental similar to those administered to group A. Animals in stresses.To the best of our knowledge, there is no study group C were neither treated with dexame-thasone nor that evaluated the relationship between stressful transported but were also infected with doses similar to conditions and the shedding pattern of Brucella melitensis those administered to group A and served as positive infection in goats.controls.Animals in group D served as the negative Therefore, this study seeks to elucidate the controls.shedding routes of experimentally-infected goats by Sampling: Blood, ocular, nasal and vaginal swabs were subjecting them to dexamethasone treatment and collected on days 0, 3, 7, 10, 14 and weekly thereafter transport stress in order to evaluate the shedding routes until day 63 post infection (pi).The blood samples were and patterns using serological tests and PCR of collected in duplicates containing both heparinized and samples collected from potential shedders.
non-heparinized samples.Dry and sterile cotton wool

Materials and Methods
swabs were used to collect the ocular, nasal and vaginal swabs.Animals: Twenty four healthy boar goats used for this experiment were housed in designated pens in the PCR assay: DNA was extracted from the heparinized Faculty of Veterinary Medicine, experimental unit, blood using Promega kit by following the manufacturer's Universiti Putra Malaysia.The animals were selected instructions after which PCR was performed using B.  stay for 10 minutes.In this test, a classical agglutination The journey commenced by 1000 hours and was reaction was considered positive for Brucella.For the terminated at 1330 hours.The vehicle travelled on a indirect ELISA, the antigen was prepared and used to typical asphalt double lane road from Serdang to coat the wells of a polystyrene plate (Nunc-Immuno Malaka and back (3°00'N, 101°40'E) Malaysia, covering plate with MaxiSorp surface).After overnight incubation a total distance of about 350km.The vehicle travelled o at 4 C, the plate was washed 3 times with the washing with an average speed of 60 km/h and had a stop-over buffer (Phosphate Buffered Saline, pH 7.4, with 0.05% Sneezing, ear biting and coughing were also observed Tween 20).200 µl of Blocking Buffer was added to to varying degrees within the first 21 days pi in groups o A and Bonly.each well and incubated at 37 C for 1hr and washed 3 times again with the washing buffer.50 µl of serum Shedding route: The animals started shedding the from samples at the dilution of 1:200 were added to the bacterium from day 7 pi, first in group B with 50% of wells and incubated for another 1hr and washed 3 times the animals shedding through the nasal route followed with the washing buffer.Horse Radish Rabbit Antiby the vaginal route (Table -1).Significant difference Goat immunoconjugate IgG Peroxidase (Nordic (P<0.05) in the shedding pattern was observed in Immunology, Netherlands) was added and incubated groups A and B when compared to group C. Group A for another hour and washed again 3 times.Then 100µl started shedding through the nasal route from day 10 pi. of 3,3,5,5-tetramethylbenzidine (TMB) One solution As the infection progressed, majority of the animals in substrate (Promega, USA) was added and incubated for group A shed the bacterium via all the routes examined 10 minutes.The reaction was stopped by the addition and relatively for a longer period with some of the of 50 µl of 2.5 M sulphuric acid.The optical density animals shedding consistently for 5 weeks.Group C, values were measured at 450 nm wavelength in an however, did not shed the bacterium via the vaginal AnthosZenyth 340 st reader (Austria).
route until day 35 pi; while groups A and B shed through this route as early as day 10 pi.Throughout the Statistical analysis: Data obtained in this study were experiment, only 1 animal from A shed the bacterium in analyzed using Statistix 9.0 software, using repeated all the 3 routes at day 21 and day 35.By day 14 pi, Measure Analysis of Variance and LSD.All-pairwise 100% of animals in groups A and B shed the bacterium comparisons was used to test the differences between at least once -while only 33% of animals in group C the groups.The criterion for statistical significance was shed the bacterium.All the animals shed the bacterium set at P < 0.05.at least once in the period covered for the experiment

Results
with the exception of the negative control group (group D) which remained negative throughout since group D General observations: Group A had a consistently was not inoculated with the infective dose.higher body temperature when compared to groups B and C. The thermal response was undulant and differed Comparison of the three shedding routes: Groups A significantly between groups.There was no significant and B shed significantly (P<0.05)higher when difference in the body temperature pattern in groups A compared to group C. 100% of the animals in group A and B, but there was a significant difference between shed the bacterium in at least 2 routes at a time in this groups A, B and C when compared to group D. A study and they shed more consistently when compared generalized inappetence was observed in all the to groups B and C (Table-2).In group A, 50% of the infected groups on day 8 pi, but more severe in group A.
animals shed concomitantly through the nasal and  identifying all the shedders.Animals in group A started In group B, however, only 33% of the animals shedding on day 10 pi, where 33% of the animals shed shed the bacterium through at least 2 routes at a time.
the bacterium.These shedders were positive with 33% of the animals shed through the nasal and ocular ELISA (ELISA been more sensitive), but negative with routes and 17% shed through nasal and ocular routes RBPT.However, the RBPT was able to identify all the while none shed through the ocular and vaginal and all shedders at day 14 pi and by day 21pi, which was the the 3 routes.None of the animals from group C shed the peak shedding period (100%) for group A, both ELISA bacterium in any 2 routes at a time throughout the and RBPT were able to detect only 17% of the animals study.Even though the ocular route was used for the in group A that were shedding the bacterium.Even infection, none of the animals from group C shed though the ability of the serological tests to identify the through this route.However, none of the animals from shedders increased over time in this study, they were all the groups shed the bacterium in any 2 routes at a not able to detect all the shedders from this group.time after day 49 and also the consistency of shedding There was no significant difference in the ability of decreased after day 49.
RBPT and ELISA in the detection of shedders and nonshedders among the group.On the other hand, PCR was Serological tests: For the RBPT, Group C tested able to detect more accurately in groups A and B than positive significantly (P<0.05) when compared to group C in all the routes with significant difference (P < groups A and B on day 14 pi, where 100% of animals in 0.05) between groups A and B compared to group C group C tested positive and the number of positives with respect to the ocular route.eventually dropped and remained constant at 50% Animals in group B however, started shedding as from day 21 to day 49 pi.60% of those in group A tested early as day 7 pi and unlike group A, peaked between positive at day 14 and the number of positives also day 7 and day 10 pi with 50% of the animals shedding.dropped to 17% from day 21 onward.50% of animals ELISA was able to detect only 25% of the shedders at from group B tested positive on day 14 and then this period, while RBPT was negative in all.dropped to 17% from day 21 onward.The indirect Subsequently, the rate of shedding dropped to 17% at ELISA (Figure -1) was able to detect the antigen earlier day 14 and ELISA was able to detect all the shedders compared to the RBPT in this study.50% of the animals while the ability of RBPT to detect the positives was in group C tested positive at day 7 pi and by day 14 pi, relatively less.We also observed that the ELISA was all the animals in this group tested positive.33% of the able to identify more number of the shedders over time.animals in Group B tested positive on day 7 pi and by day 42 pi, 100% (6/6) tested positive and remained Animals from group C shed less of the bacterium positive throughout the study.Only 17% of the animals compared to groups A and B, as expected.By day 14, in group A tested positive on day 7 pi but 100% by day none of the animals in group C was shedding via the 49 pi and remained positive throughout the study.
routes examined even though some serologically tested There was a significant difference (P<0.05) in the positive with both ELISA and RBPT.antibody titres among the treatment groups from day 7 Polymerase Chain Reaction (PCR) was carried through day 42 pi.out using DNA extracted from the blood samples of the animals and this test detected positive animals earlier Relationship between the shedding routes and the than the serological tests.PCR detected 100% of the serological response: In this study, the stressed animals animals in group C and 50% in group B as early as day 3 showed increased shedding of the bacterium and in at pi.By day 7 pi, blood samples from all the infected in the stressed groups vs. the control animals (group C animals tested positive and thereafter, most of the -infected, but not subjected to prior stress) further animals were negative.However, group D (the uninfected implies that stress could induce pathogen redistribution within the system of the infected animals and thereby control group) remained negative throughout the study.constituting a high public health risk.This is typified Discussion by the earlier onset of shedding via the vaginal route as The shedding of B. melitensis by domesticated observed in the stressed groups (groups A and B), when animals poses a very serious public health risk.Infected compared to the unstressed group (group C), despite animals are often the source of human infection with the use of intraocular route for the inoculation purpose.brucellosis, especially domesticated animals and the The peak shedding observed between days 7 and 10 pi control of this disease in humans is only achievable in group B showed the critical period at which the through the effective control of the disease in animals, transported animals could shed; this period should be hence its public health significance.Most Control taken into account while devising the control strategies.The shedding observed in group A showed programmes for Brucellosis often rely on serological that chronically-stressed or immunosuppressed goats tests for screening and/or identification of infected could continually shed the bacterium for considerably animals without any emphasis on the detection of a longer period as observed in diseases such as Q-fever apparently animals that are shedding the bacterium.
and enteric infections [19,22].This study evaluates the shedding routes and the Our study also showed that the shedding rate in diagnostic potential of analyzing more than one group C was relatively lower and inconsistent (Table -

1). biological sample in experimentally-infected
The increase in the number of shedding routes in the (dexamethasone-treated or transport-stressed) goats as stressed animals, as reported in this study, further evaluated for Coxiella burnetti infection in the earlier demonstrates the important role of stress in inducing studies [19,20].
not only the bacterial redistribution in vivo but also in All the infected animals in this study shed the the disease transmission.bacterium at least once via one of the 3 routes In this study, the two serological techniques employed investigated, the earliest being the nasal route from the showed that stress induced either by dexamethasone or transport-stressed group (group B) at day 7 pi while by transport is capable of causing immune dysreguthose belonging to the dexamethasone-treated group lation [13, 24], an affect that possibly can lead to less (group A) shed more consistently in all the routes seroconvertion observed in the stressed groups examined and for a longer period starting from day 10 showing only 50% of the animals being seropositive pi when compared to animals that were infected -while group C showed a 100% (6/6) seroconvertion without any prior stress-inducing treatments (group C).
within the same period.The use of the serological This result is in accord with earlier studies [21,22,23] techniques to identify potential shedders, especially in which reported that stress factors are linked to stressed goats, were not encouraging as our study increased pathogen carriage, increased disease showed that RBPT identified all the animals (100%) susceptibility, and possible pathogen shedding.Thus, shedding the bacterium in group C whereas only 66% stress factors can lead to immune dysregulation [24].and 50% of the animals from groups A and B tested There is evidence to show that immunosuppression positive, respectively.The indirect ELISA used in this could account for severe lesions being encountered in study was able to detect positive animals at an earlier the respiratory tract in diseases such as peste des petits time when compared to the RBPT but it could not ruminants [13], and this may be one of the reasons why identify all the shedders in the course of this study until we observed the dominance of the nasal route in the day 35.The reduced and inconsistent serological shedding pattern in our study.The proximity and the responses observed in groups A and B as detected with linkage of the ocular and the nasal routes could also indirect ELISA and RBPT could possibly be assigned explain the dominance of the nasal route in shedding to stress-induced immune dysregulation [13].the bacteria.
The PCR performed using the DNA extracted The earlier onset and prolonged shedding of the from the blood samples identified the infected animals bacterium by the stressed and infected animals in this as early as day 3 pi, and on this day all the animals in study further confirmed that the combination of group C tested positive 50% from group B and none infection and stress could pose a very serious risk to the from group A tested positive.By day 7 pi, all the blood public health.Our finding also indicates that there is a samples of the animals infected in this study tested high potential for the spread of this infection to humans positive with PCR.Subsequently, the animals tested who are involved in small ruminant husbandry practices, positive only sporadically.This could be attributed to especially in the tropics.In agreement with our the fact that the bacterium could have migrated from findings, earlier studies [25,26] reported that Brucella the blood and get trapped within the macrophages and infected animals could serve as sources of infection to non-professional phagocytes [27].Furthermore, it was other non-infected animals as well as the human speculated that the inconsistency in the positive results populace.The relatively increased shedding of the could be due to re-infection originating from intermittent bacterium through the nasal, ocular and vaginal routes shedders.Our study provides vital information on the role

7
broth containing 10 CFU of B. melitensis strain 183 The handling, loading and transportation of the using Promega Genomic DNA purification kit (Promega, experimental goats were carried out humanely in Germany).accordance with the guidelines governing animal Sera were also harvested from the nontransport welfare by road[16].A standard Bedford, o heparinized blood and stored at -20 C until used for open-roof (Ford, UK), vehicle was used for the RBPT and ELISA.For the RBPT, a drop from each transportation.The floor of the van was provided with serum sample was laid on a plate and a drop of the beddings (wood shavings) for secure footing.The 2 RBPT reagent was added, mixed well and allowed to goats were stocked at a rate of 0.2 m per animal[17].

Table- 2 .
Comparison of the relationship between the 3 shedding routes , V: Vagina and O: Ocular route vaginal routes, 33% through nasal and ocular routes, least 2 out of the 3 routes investigated, but the serological 50% through ocular and vaginal and 17% shed twice tests (RBPT and iELISA) were not consistent in via all the 3 routes.

Figure- 1 .Figure- 2 .
Figure-1.The pattern of antibody response to experimental B. melitensis infection in group A : 1, B : 2 and C : 3; in percentage of positivity . (2008) Development of a real-time PCR assay for Conclusion diagnosis of Brucella melitensis infection in sheep.
The experimental design and animal 15minutes before adding the master mix as described treatment was in compliance with the humane methods above.The negative control used was the master mix recommended by the Research Ethics Committee, without DNA template and the external positive Faculty of Veterinary Medicine, Universiti Putra control was DNA templates extracted from a Brucella Malaysia (AUP No: 12R159).
earlier.Briefly, 2µl of DNA Zol was added to 2µl of the colonies in Eppendorf tubes and allowed to stay for Ethical approval:

Table - 1
. PCR result for Nasal, Ocular and Vaginal swabs that were tested for B. melitensis.
University Putra Malaysia Institutional repository, 2008 8. Ayo, J.O. and Oladele, S.B. (1996) Natural antioxidants and of stress in modulating the pattern and routes of their potential uses in prophylactics and therapy of disease shedding of B. melitensis in dexamethasone-treated conditions.W. Af.J. Pharmacol.Drug Res., 12: 69-76.and transport-stressed goats.Our significant findings 9.