Seroprevalence, Knowledge, Attitudes and Practices of Porcine Brucellosis in Mifi Division, West Region Cameroon

Received 01 Feb, 2021

Accepted 10 May, 2021

Published 10 Jun, 2021

AbstractBackground and Objectives: Brucellosis is a devastating and neglected disease that affects animal and human health worldwide. The epidemiology and knowledge, attitudes, and practices (KAP) for this disease in pigs were observed. Materials and Methods: It is in this context that a cross-sectional study was carried out to determine the seroprevalence and KAP of porcine brucellosis in the Mifi Division of West Region Cameroon. A total of 456 sera were collected from slaughtered pigs in the Bafoussam pig abattoir. Data on pigs and their environment was collected using a questionnaire administered to 117 professionals in the pig sector to assess their KAP for porcine brucellosis and level of awareness of porcine brucellosis amongst pig professionals. All sera were submitted to the Rose Bengal Plate Test (RBPT) and competitive ELISA (COMPELISA). Results: The results showed seroprevalence rates of 6.35% and 1.53% in the RBPT and COMPELISA tests respectively. Seroprevalence rates for sows were higher (2.82%) compared to boars (0.40%). With respect to age, the most sensitive age class was between 12 -36 months with a seroprevalence of 1.74% followed by animal between 6-12 months (1.47%). There was a significant difference in the seroprevalence of large white hybrid breed (2.13%) compared to local (1.45%) and Duroc hybrid (1.25%). There was no relationship between seroprevalence observed and symptoms related to porcine brucellosis. Minority 2.9 % of the surveyed population had knowledge of animal brucellosis and mode of transmission with 0.8% of them being aware of porcine brucellosis. None of the respondents knew that brucellosis was a zoonosis. Conclusion: Hence, Factors such as ignorance of the hazards and modes of transmission of zoonotic brucellosis by pig professionals have shown an increased risk factor for brucellosis in pigs and the exposure of pig professionals to the infection.

INTRODUCTION

Cameroon possesses the highest pig population in the Central African sub-region with an average estimated population of 1 963 355 headcounts. Despite this potential, production remains highly insufficient and the consumption of pork per individual remains very low (2.98 Kg) instead of 5 Kg recommended per habitant per year¹. Several constraints such as poor organisation of the pig sector, lack of adequate genetic resource, feeding and high incidence of diseases². Although, African swine fever is the most important pig disease^2,3, there are many other devastating and neglected diseases among which porcine brucellosis rank as a limiting factor for the development of the pig sector in Cameroon⁴. Porcine brucellosis is a highly contagious zoonotic disease caused mainly by Brucella suis and occasionally by Brucella abortus and Brucella melitensis⁵. It has been associated with huge economic losses due to factors in sows related to decreased farrowing percentage and litter sizes, delayed farrowing, culling for infertility, cost of treatment, abortions, stillbirths, birth of mixed live but weak and dead piglets, orchitis in boars and loss of man-hours in infected communities^6,7. Apart from its impact on animal health, it causes public health problems⁸. Zoonotic brucellosis also causes reproductive, nervous and other clinical disorders in humans⁹, who are usually occupationally exposed to infected pigs¹⁰. Risk of transmission from animal to man is important due to husbandry practices, feeding habits and practices of the concerned population¹¹. Brucellosis is an important notifiable disease of man and animals worldwide. In Cameroon, epidemiological data are mostly found on bovine brucellosis^12,13. Despite the fact that there are vulnerable communities and populations at risk including livestock and livestock professionals little is known on the epidemiology of porcine brucellosis. Therefore, this study was carried out to contribute knowledge on the epidemiology of porcine brucellosis in Cameroon. More specifically to determine the effect of breed, sex and age on the seroprevalence of porcine brucellosis in Mifi Division of West Region Cameroon, Relationship between symptoms and seroprevalence of porcine brucellosis in Mifi Division, knowledge, attitudes and practices related to porcine brucellosis pig professionals in Mifi Division of West Region Cameroon.

MATERIALS AND METHODS

Study area: This cross-sectional study was carried out from February to June 2019, in Bafoussam (Latitude 5^o25’0”-5^o35’0” N and longitude 10^o20’0”-10^o35’0” E) in the Mifi Division of the West Region of Cameroon. The west region is located in the Equato-Guinean highland with an average annual precipitation of 1600-2000 mm in the rainy season which runs from around March to November. Mean temperature ranges from 23-26°C with a maximum of up to 32°C. The region is the principal pig production zone in the country.

Selection of animal for the study: Selection of pigs for the study was done in the Bafoussam municipal abattoir during the study period using previously described systematic random sampling technique¹⁴. An individual prevalence rate of 50% was used to estimate the minimum sample size (384 pigs) as described by Thrusfield¹⁵ then rounded up to 456 pigs. Briefly, about 20% of 40–60 pigs slaughtered daily in the abattoir was randomly selected each day, except on Saturdays and Sundays were included in the study. Based on a calculated sampling fraction of five (every fifth animal was sampled) for daily use, the first animal was selected by picking one animal by random generation method of the first five animals on the slaughter chain. Thereafter, every fifth animal (adding 5 to previous picked number) was chosen till the sample size was achieved. Information related to the breed, sex, age and body condition score of the animal were noted. Estimation of ages was done by dental inspection while the breeds of the animals were obtained as previously described by Thrusfield¹⁵. The body condition score was done by assessing the general appearance and palpation of the lumbar region of the animal on a scale of 1 to 5 and further classed into 3 categories: 1–2 (poor), 3 (good) and 4–5 (very good) as previously described by Fitzgerald¹⁶.

Blood sampling and laboratory analysis: Apart from procedural restraining manipulations for safety purposes and jugular venipuncture for blood sampling, the animals were not subjected to suffering. Blood samples (≥5 ml) were collected using sterile vacutainer from the chosen animals on the chain before slaughter¹⁷. Serum samples were extracted from collected blood and stored at - 20°C until laboratory analysis at the Veterinary Research Laboratory of the Institute of Agricultural Research for Development, Wakwa Regional Center, Ngaoundere, Cameroon.

Serological test for porcine brucellosis: After screening all 456 pig serum samples with Rose Bengal Plate test (RBPT), Enzyme-Linked Immunosorbent Assay (ELISA) was performed on samples to detect anti-brucella antibodies. Each batch of test was included with a positive control and a negative control. A subject was seropositive when the serum tested positive to RBPT and or ELISA¹⁷.

Rose Bengal Plate Test RBPT was performed as described by Yagupsky et al.¹⁸. Briefly, the sera and antigen were brought to room temperature before use. Equal volumes (30 μL) of standardized B. abortus antigen Weybridge strain 99 and test serum were mixed thoroughly and rotated on a glass plate using a stick applicator, and the plate was rocked for 4 min. The appearance of agglutination, recorded as positive, within 1 min was scored 4+ (++++) and between 1 and 4 min was scored 1+ to 3+ (+, + +, and + + +) according to the different degrees of agglutination. The absence of agglutination within 4 min was regarded as negative (-).

Detection of brucellosis antibodies in pigs, Commercial indirect multispecies ELISA (i-ELISA) (COMPELISA, Apha Scientific, England) for the detection of antibrucella (B. arbortus, B. melitensis and B. Suis) antibodies in the pigs serum was performed according to the manufacturer’s instructions and essentially as described by Geresu and Kassa¹⁹. The test was conducted in 96-well polystyrene plate that was precoated with purified Brucella abortus lipopolysaccharide (LPS) antigen. An anti-multi-species-IgG horseradish peroxidase (HRP) was used as conjugate as described by Howell²⁰. The substrate solution (TMB + DMSO + H₂O₂) was added after washing to eliminate excess conjugate. The coloration of antigen-antibody conjugate-peroxidase complex formed depended on the quantity of anti-Brucella antibodies that was present in the specimen tested. Thus, in the presence of antibodies, a blue solution appeared which became yellow after addition of the stop solution, while in the absence of antibodies, no coloration appeared. The optical density (OD) of the well was read at 450 nm by an automatic micro plate reader and for each sample S/P% was calculated²¹ as follows:

$$ \frac{s}{p} \%=\frac{\text { ODsample }-O D n c}{O D p c-O D n c} * 100 $$

where ODsample, ODnc, and ODpc are the readings of optical densities for the sample, negative control, and positive control, respectively. The samples were classified as positive if S/P% ≥120%, negative if S/P%≤110%, and doubtful if 110%<S/P%<120%. Also, the fact that ODpc >0.350 and ODpc/ODnc >3 indicated that the tests were working properly.

The seroprevalence and confidence interval¹⁸ were calculated using the formulae below:

Seroprevalence = (Number of brucella positive animals/Total number of animals tested)/ 100

$$ \text { Confidence interval, } \mathrm{CI}=p \pm z^{2} \sqrt{\frac{p(1-p)}{n}}, z=1.96 \text { at } 95 \% \mathrm{CI} $$

Assessment of knowledge, attitudes and practices of pig brucellosis by pig professionals: In this study, the assessment of the KAP for human exposure to porcine brucellosis and its level of awareness amongst pig professionals was done among staff and workers of Casa Blanca market abattoir in Bafoussam. The data were obtained using direct interview through a semi-structured questionnaire. KAP related to the lifestyle of the respondent, his/her sex, age, level of education, formal training in the pig profession, interaction with pigs and other animals, protective measures in the exercise of his profession, his reproductive health since he/she became active in the pig sector, preventive measures at work, knowledge of animal brucellosis in general and porcine brucellosis in particular. A total of 117 individuals including 48 pigs farmers and 69 abattoir personnel (Butchers, carcass cleaner, transporters, pork roasters and veterinary agents) were surveyed

Data analysis: Data collected from the questionnaire survey was recorded and analysed using Sphinx plus2 version 4.0. Descriptive statistics SPSS software version 21 was used to analyze the data obtained. Univariate analysis for correlation between potential occurrence of reproductive disorders and Brucella status and the various test performed was carried out using Chi-square test and the test level for statistical significance was set at 5% (95% level of confidence, p<0.05)¹⁹.

RESULTS

Combined results of Rose Bengal Plate test and Competitive enzyme linked immunosorbent assay among pigs in Bafoussam: The study showed that of 456 samples tested, 29 (6.35%) and 07 (1.53%) were positive to Rose Bengal plate test and competitive ELISA test respectively in Table 1.

Table 1: Seroprevalence of porcine brucellosis in function of serological test used
Type of test	Positive cases (n)	Prevalence (% [95% CI])
RBPT (+)	29	6.35 [4.46- 8.98]
RBPT (+) and COMPELISA (+)	07	1.15 [0.75- 03.13]
RBPT (+) and COMPELISA (-)	19	4.17 [2.68- 6.42]
(+): positive; (-): negative; RBPT: Rose Bengal Plate Test; COMPELISA: Competitive Enzyme- Linked Immunosorbent Assay; n: Number of positive cases

Seroprevalence of pig brucellosis with respect to sex, age and breed: The study showed that seroprevalence of porcine brucellosis had a significant increase in sows (2.82%), pigs of atleast 12months (1.74%) and large white breed(2.13%) (p< 0.05).

Table 2: Seroprevalence of pig brucellosis with respect to sex, age and breed
Parameter	Variable	Number (positive cases)	Prevalence (% [95% CI])	Odd ratio (% [95% CI])	P-value	Chi-square
Sex	Sows	213(06)	2.82 [1.30-6.01]	2.90 [1.31 - 6.39]	3,20E-100	503,952
	Boars	243(01)	0.40 [0.07- 2.23]	0.41 [0.07- 2.28]	(P<0,0001)
Age (Months)	[6-12]	341(05)	1.47 [0.63- 3.39]	1.49 [0.63- 3.50]	1,03E-95	470,783
	[12-36]	115(02)	1.74 [0.48- 6.12]	1.77 [0.48- 6.52]	(P<0,0001)
Breed	Duroc Hybrid	80( 01)	1.25 [0.22- 6.75]	1.27 [ 0.22- 7.23]	1,45E-93	472,663
	Large White hybrid	94(02)	2.13 [0.59- 7.43]	2.17 [0.59- 8.03]	(P<0,0001)
	Local	275(04)	1.45 [0.57- 3.68]	1.48 [0.57- 3.82]
	Naima	05 (0)	0	0
	Land race hybrid	02 (0)	0	0
Total		456 (07)	1. 54[0.75- 3.13]	1.56 [0.75- 3.24]
( ): number positive case; [ ] : 95% confidence interval; P: Pearson value

Relationship between symptoms observed and seroprevalence of pig brucellosis: Majority of sampled animals (94.5%) never manifested any clinical symptoms associated to pig brucellosis but were seropositive to RBTP and COMPELISA brucellosis test whereas those that manifested symptoms associated to pig brucellosis were seronegative to RBTP and COMPELISA brucellosis tests.

Table 3: Relationship between symptoms observed and seroprevalence of pig brucellosis
Symptom	Number tested	RBPT	COMELISA	Seroprevalence (%)
Mastitis	14	00	00	0.0
Orchitis	05	00	00	0.0
Vaginitis	5	00	00	0.0
Arthritis	1	00	00	0.0
Testicular Dissymmetry	1	00	00	0.0
No symptom	429	29	07	1.53
Total	456	29	07	1.53

Knowledge of brucellosis, attitudes and preventive measures practiced amongst pig farmers and professionals: The KAP study has involved 117 individuals: 48 pig farmers and 69 personnel involved in the slaughtering and transformation chain (Butchers, carcass cleaner, transporters, pork roasters and veterinary agents, Table 4).

Table 4: Knowledge of brucellosis, attitudes and preventive measures practiced amongst pig farmers and professionals
	Characteristics	Frequency	Percentage
Formal Training	Yes	23	20
	No	94	80
System of reading	intensive	0	00
	Semi-intensive	48	100
	Extensive	0	00
Activity	Primary activity	39	81.3
	Secondary Activity	9	18.
Health Monitoring	Yes	20	41.7
	No	28	57.1
Pork consumption	yes	44	91.7
	No	4	8.3
Pathologies encountered	Digestive	19	32.2
	Reproductive	22	37.3
	Locomotory	1	1.7
	Dermal	5	8.5
	Respiratory	12	20.3
Management of sick animals or carcase(117)	Veterinary inspection	8	6.8
	Bury	59	50.8
	Consumption	25	21.0
	Sell	24	20.3
Biosecurity measures against brucellosis	Wearing of boots	46	39.5
	Work cloth	44	38.2
	Cleaning of working premises	74	63.3
Knowledge on Brucellosis and its hazards	yes	4	2.9
	No	113	97.1
Mode of transmisison	yes	1	0.8
	No	116	99.2
Consumption of pork	Flesh	73	62.7
	Viscera	44	37.3
Preventive Measures against Brucellosis	Spare clothing	56	48.5
	Wearing of boot	3	2.6
	Wearing of blouse	15	13.4
N= 117

Only 20% of the farmers surveyed had received training in pig farming; the system practised is semi-intensive with generally less than 10 pigs (75.5%). Livestock farming represented the main activity for 81.3% of respondents, while health monitoring is carried out on 41.7% of farms. Reproductive problems (37.3%) and digestive problems (32.2%) are the most common on the farms whose farmers participated in this survey. No specific protective measures are taken by livestock owners when assisting during farrowing or managing abortions; corpses are buried (50.8%), consumed (22%) or sold (20.3%); less than 10% of livestock owners use a veterinarian. The main biosecurity measures in the performance of their tasks are the wearing of boots (39.5%) and work clothes (38.2%), and cleaning is carried out on a daily basis using running water (63.3%), with disinfection being practised by half of the individuals surveyed. Less than 5% of the livestock owners surveyed knew of brucellosis, and only one knew that it is a zoonosis.

Minority (2.9%) of the surveyed population had knowledge of animal brucellosis and the mode of transmission with 0.8% of them being aware of brucellosis in pigs. None of the respondents knew that brucellosis was a zoonosis. Pork is consumed by 97.1% of respondents, who prefer the flesh (62.7%) to the viscera (37.3%). Among the means of protection used in the conduct of their activity, we can mainly cite spare clothing (48.5%), boots (27.6%) and blouse (13.4%). Apart from the sensation of testicular warmth/pain, none of the proposed reproductive problems were noted among the respondents.

DISCUSSION

This study had as aim to determine the seroprevalence and to assess the knowledge, attitudes and practices of pig professionals towards porcine brucellosis in Bafoussam town of West Region Cameroon. Serological test using Rose Bengal Plate Test (RBPT) and competitive ELISA (COMPELISA) revealed serological prevalences of 6.35 and 1.15% respectively. The difference in prevalence obtained in the study was surely due to the difference in sensitivity and specificity of the two serological tests used^13,24. A combination between RBPT and COMPELISA revealed a prevalence of 1.53%. This is a disease with low prevalence so chances of having a seropositive case are very low for a small sample size. This study used RBPT and competitive ELISA in combination to minimize the possible measurement of false positive errors and revealed that porcine brucellosis is a real pig and human health problem in piggery structures in Bafoussam. In fact, RBPT is internationally recommended for screening of large numbers of sera while competitive ELISA offers the highest sensitivity and specificity of all currently available serological tests^21,22.

The seroprevalence of porcine brucellosis was significantly influenced by sex, age and breed. Sows were more sensitive (2.82%) to porcine brucellosis compared to boars (0.40%). This finding has some similarity with previous reports by Praud et al.²³, Kebeta et al.²⁴, Bayemi et al.²⁵, Scolamacchia et al.²⁶, Onunkwo et al.²⁷, Mazeri et al.²⁸, Ram et al.²⁹, with higher seropositivity amongst sows than in boars in Ethiopia and associated the difference to the fact that the female reproductive tract is a potential reservoir for Brucella spp to propagate. The finding is contradictory with previous report by Godfroid et al.¹⁷, who reported a highly significant seroprevalence (1.52%) in improved Berkshire hybrid from Littoral, North- West, West, South Regions of Cameroon and did not observe a difference in Seropositivity due to sex. Bovine brucellosis is widespread in Cameroon including the West Regions^14,15,30,31 and its existence seemed to have favoured the occurrence of porcine brucellosis in the West region¹⁷. Similar findings had showed that brucellosis in pigs may occur in regions where brucellosis is endemic in domestic ruminants³².

Age had a significant influence (p<0.05) on the prevalence of porcine brucellosis in this region. This finding is in line with that reported by Godfroid et al.^.17 who noticed a prevalence of 1.85% in pig of at least 12 months old and related it to the fact that old pigs (≥12 months) had increased odds of being seropositive reactors than younger pigs and older animal are potentially exposed longer to the disease than younger animals^17,29. Aging and high parity have been observed to significantly increase brucellosis seroprevalence and animals become more sensitive to brucella infection at the reproductive age³⁰. Large white hybrid was more susceptible to brucella infection than the local and Duroc hybrids breed. The finding has some similarity with previous findings by Hegazy³¹, who reported higher seropositivity among improved breeds (Pietrain, Landrace and Duroc hybrids) but did not observe differences in seropositivity due to age. The exclusion of weaned piglets and pigs less than six months, due to poor reactions to various serological tests for brucellosis^32,33 as well as the higher exploitation of young animals and other management practices in farms might have played additional roles in the different seropositivity results due to breed in the study.

There was no relationship between symptoms related to brucellosis and the seroprevalence observed. All seropositive cases were recorded from animals that did not present any signs/ symptoms nor lesions of porcine brucellosis. This might be due to the fact that, these symptoms observed were not pathognomonic to porcine brucellosis. Other diseases manifesting similar symptoms such Aujeszky's disease, leptospirosis, Erysipela, salmonellosis, classical swine fever, porcine parvovirus infection³⁴ are possible differential diagnosis in this animal population.

Brucellosis is a zoonotic disease that can be transmitted to humans mainly by domestic animals such as cattle, pigs, goats and sheep³⁵. It appears to be an occupational disease particularly affecting herders, agricultural workers, veterinarians, butchers and dairy workers³³. In this study, very high levels of ignorant of pig professionals / handlers are aware of porcine brucellosis and its hazard (98.3%), risk factors and modes of transmission (99.2%) of zoonotic brucellosis. Several reports focusing on bovine brucellosis in Cameroon, Nigeria, Tanzania and Egypt have highlighted higher seroprevalence rates of brucellosis among livestock professionals (e.g. uneducated animal handlers, butchers, herdsmen, livestock owners and traders) less informed about zoonotic brucellosis than in more knowledgeable professionals (e.g. educated animal handlers, veterinarians and para-veterinarians) and persons with short exposure time to animal products and good personal hygiene practices (e.g administrative staff, animal owners, traders of animals and animal products who are knowledgeable)^35,36,36. Ignorance of porcine brucellosis, lack of traceability of pigs destined for slaughter and abattoir environments conducive for the survival of Brucella species which constitute major factors for human exposure to zoonotic infection^8,10,13, were noted in this study. Other potential factors associated with human brucellosis seroprevalence in livestock professional groups include consuming poorly roasted pork, handling animal foetuses and aborted animals, occupational exposure for at least 5 years, little or no knowledge of brucellosis, contact with livestock and religious practices⁸. Similar KAP situations have been described among pig professionals / handlers for brucellosis as well as the need to improve anti-brucellosis education and the routine screening of animals for swine brucellosis².

CONCLUSION

This study reported on the seroprevalence and potential risk factors of porcine brucellosis as well as level of awareness of porcine brucellosis amongst pig professionals in Bafoussam (Mifi Division) of West Region Cameroon.The findings show that exotic breeds, sows and pigs older 12 months are more susceptible to porcine brucellosis than local breeds, boars and younger pigs in Bafoussam. There was no relationship between symptoms related to porcine brucellosis and brucella positive status in Bafoussam. Ignorance of porcine brucellosis in farms and abattoir environments conducive for the survival of Brucella species constituted the major potential risk factors for human exposure to the brucella infection. The implementation of bacteriological examination for the presence of cross-reacting bacteria and isolation of these bacteria would be essential data for proper interpretation of porcine brucellosis serological test results.

ACKNOWLEDGMENT

Our profound gratitude goes to the Dschang School of Agricultural and Environmental Sciences (DSAES) of the University of Dschang and the Institute of Agricultural Research for Development, IRAD Wakwa, Cameroon for permitting us to use their laboratories.

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