Study on prevalence, clinical presentation, and associated bacterial pathogens of goat mastitis in Bauchi, Plateau, and Edo states, Nigeria

Aim: This study aimed to estimate the prevalence and clinical presentations of different forms of mastitis and mastitis-causing pathogens in lactating goats in Bauchi, Plateau, and Edo states, Nigeria. Materials and Methods: A total of 500 quarters from 250 lactating goats of Red Sokoto and West African Dwarf breeds during the lactation period were clinically examined. Clinical mastitis was detected by gross signs of udder infection during physical examination and abnormal milk, whereas subclinical mastitis (SCM) was recognized using California mastitis test. The bacterial pathogens were identified by morphology, hemolysis, gram staining, and biochemical tests such as catalase, oxidase, coagulase, reaction on sulfite, indole, and motile medium, and fermentation of sugars. Results: The overall prevalence of mastitis in goats was found to be 101 (40.4%), of which 8% (20/250) were clinical, and 32.4% (81/250) were SCM cases. The quarter level prevalence was 29.4% (145/493), comprising 5.9% (29/493) clinical and 23.2% (116/493) subclinical forms of mastitis. In addition, 1.4% (7/500) of teats were found to be blind on the clinical examination of the udder and teat. Several regional inflammatory reactions and abnormalities in milk were found in 69% and 100% of the cases, respectively. Moreover, some indications of generalized signs such as fever, reduction in appetite, increase in respiration, and pulse rate per minute were recorded in 100%, 75%, 85% and 80% of the cases, respectively. The predominant bacterial isolates recovered were Staphylococcus aureus (20.0%), followed by Escherichia coli (15.5%) and Streptococcus agalactiae (11.0%), and the least isolated microorganisms (≤6%) were bacteria of different species including Staphylococcus auricularis, Staphylococcus caprae, Staphylococcus chromogenes, Staphylococcus epidermidis, Staphylococcus hyicus, Staphylococcus xylosus, Staphylococcus lentus, Streptococcus dysgalactiae, Streptococcus pluranimalium, Streptococcus uberis, Streptococcus pneumoniae, Streptococcus ruminatorum, Streptococcus suis, Micrococcus luteus, Enterobacter cloacae, Proteus vulgaris, Klebsiella oxytoca, Klebsiella pneumoniae, Morganella morganii, Salmonella Typhimurium, Citrobacter freundii, Pseudomonas aeruginosa, Acinetobacter rudis, Acinetobacter haemolyticus, and Bacillus cereus. Conclusion: Mastitis continues to be recognized as one of the important health issues and leads to major economic losses to the dairy goats caused by many bacterial pathogens, and the effective measures need to be taken to control the disease.


Introduction
Small ruminants play a very significant role in the socioeconomic and nutritional requirements of most countries. In Nigeria, many families keep goats as a source of complementary income [1]. Within the past 7 years, the recent FAO statistics [2] puts the number of goats in the world at 861.9 million with 53.8 million goats in Nigeria alone. This represents 6.24% of the total goat population in the world [2]. Goat production and productivity and producers' benefits are far below expectations due to disease, malnutrition, low genetic potential, and poor management practices. Mastitis and other infectious diseases are the major constraints to goat's production in Nigeria [3]. Among infectious diseases, mastitis is one of the major diseases affecting dairy goat productivity [4,5].
Mastitis is a parenchymal inflammation of the mammary gland, characterized by physical, chemical, and usually bacteriological changes in milk and pathological changes in glandular tissues [6,7]. In general, mastitis occurs in two forms which include clinical (overt) which was subdivided into serous, catarrhal, purulent-catarrhal, and gangrenous and subclinical (hidden) [8,9]. Clinical mastitis (CM) is characterized by sudden onset of swelling and redness of udder, pain, and reduced and altered milk secretion from the affected quarters. The milk may contain clots or flakes or become watery in consistency accompanied by fever, depression and anorexia [10]. Mastitis is a major cause of substantial economic losses in dairy goat's flocks. Economic losses are due to decreased milk yield, changes in milk quality and composition, increased cost of treatment, increased mortality of kids, and potentially premature culling of small ruminants [11,12].
The predominant pathogens for mastitis throughout the world include Staphylococcus, Streptococcus, and coliform species [13]. In Nigeria, several etiological agents of mastitis in small ruminants have been reported, such as Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus agalactiae, Streptococcus dysgalactiae, Klebsiella pneumoniae, Escherichia coli, Salmonella Typhimurium, Shigella flexneri, Proteus vulgaris, Proteus mirabilis, Bacillus spp., Corynebacterium pseudotuberculosis, and Pseudomonas aeruginosa [14,15]. Predisposing factors such as poor management and hygiene, teat injuries, and wrong milking methods are known to play an important role in the invading of bacterial pathogen in the glands and occurrence of disease [16].
This study aimed to estimate the prevalence and clinical presentations of different forms of mastitis and mastitis-causing pathogens in lactating goats in Bauchi, Plateau, and Edo states, Nigeria.

Ethical approval
All the animals in this study were treated according to the ethical standard of Moscow State Academy of Veterinary Medicine and Biotechnology -MBA named after K. I. Skryabin. The experiment was carried out in accordance with the guidelines laid down by the International Animal Ethics Committee and in accordance with Local Laws and Regulations.

Study area
This study was carried out in 54 herds selected (at random from three Nigerian States) (Figure-1), namely: Bauchi state is one of the six states of the northeastern region of Nigeria. It is located between latitudes 9°3' and 12°3' and longitude 8°5' and 11° east.
Bauchi state occupies a total land area of 49,119 km 2 representing about 5.3% of Nigeria's total landmass. Bauchi state has an estimated population of 4,653,066 people with 20 local government area councils [17]. The state has a small ruminant population of 2.8 million sheep and 3.4 million goats [18].
Plateau state is located in Nigeria's Middle Belt. It is located between latitude 08°24'N and longitude 008°32' and 010°38' east. Plateau state occupies a total land area of 26,899 km 2 . The state has an estimated population of 3,206,531 people with 17 local government area councils [17]. The state has a small ruminant population of 964,188 sheep and 1,865,805 goats [19].
Edo state is situated in the mid-southwest of Nigeria. It is located between latitude 6.5438° N and longitude 5.8987° E. The state covers 17,802 km 2 . Edo State has estimated population of 3,233,366 people with 18 local government area councils [17].

Study animals
The animals were lactating goats including Red Sokoto and West African Dwarf breeds, managed under extensive or semi-intensive system.

Study design and sample size determination
A cross-sectional study was used to screen mastitis in lactating goats. The sample size was determined according to Thrusfield [20] formula with an expected prevalence of 12.65% [15] and 95% statistical confidence level. Accordingly, the sample size of lactating goats was determined to be 170 based on the formula; however, for the purpose of this study, a total of 250 lactating goats, 125 each from extensive and semi-intensive system, were included in the study. In general, 54 herds, of which 36 were small-holders (owning ˂5 lactating goats) and 18 from medium-sized (owned ≥5 lactating goats) owned by private individual owners, were included in the study. The basis for sampling was the production system (extensive or semi-intensive) practiced by individual farmers in the study area. Extensive system was defined as production system where goats are left to wander and graze during the day and are enclosed during the night, whereas in semi-intensive system, the goats are limited to grazing during the day, fed with dry legume hay (pasture) and supplemented with a local concentrate popularly known as dusa-massara (maize offal), and also enclosed during the night. A general study was carried out, then systemic clinical and special studies (inspection and palpation of the udder, forestriping test and microscopic evaluation of milk), and laboratory test of the milk (culturing, isolation and biochemical identification of the isolated microorganisms).

Clinical examination
The udders were carefully inspected followed by thorough palpation to detect temperature, possible fibrosis, inflammatory swellings, visible injury, tick infestation, atrophy of the tissue, and swelling of supramammary lymph nodes. Rectal temperature Available at www.veterinaryworld.org/Vol.12/May-2019/3.pdf was measured by a digital clinical thermometer (Model ICO Technology "mini color," Barcelona, Spain; range, 32-43.9°C; accuracy, 0.1°C, pulse rate (PR) was taken from the saphenous artery, whereas the number of inhalations and exhalations during 60 s indicated the respiration rate. During the study, 20 clinically affected goats (with 29 udders: Both halves -20 and one halve -9) were studied in detail. The clinical cases of mastitis observed were classified as serous, catarrhal, and purulent-catarrhal [21] and defined as follows.

Serous mastitis
Warm, swollen, hyperemic, painful mammary gland with abnormalities of the milk. Affected animals may show signs of depression, fever, and recumbency but are not usually as severely affected as those with peracute mastitis [22]. Supramammary lymph nodes were 1.5-2 times enlarged and painful to palpation.

Catarrhal or peracute mastitis
Less apparent inflammation of the mammary gland, but with abnormalities in milk. Affected animals may show signs of depression, fever, and dehydration and frequently become recumbent [23]. Supramammary lymph nodes were enlarged and painful to touch.

Purulent-catarrhal
Depression and fever. The affected udders were swollen, hard, painful, and hot, with a grossly enlarged teat. Bluish-reddish tinge and spotty hemorrhages may be visible on the skin. Supramammary lymph nodes were enlarged and painful. The secretion from the affected udder is usually thick with foul-smelling yellow-green pus [21].

Diagnosis of subclinical mastitis (SCM)
The California mastitis test (CMT) was used to detect the presence of SCM. This screening test was performed according to the procedure given for mastitis by Quinn et al. [24]. The result was scored as 0, +1, +2, or +3 depending on the intensity of reaction. Samples with CMT result score of 0 and +1 were considered as negative, while those with a score of +2 or +3 were taken as positive.

Milk sample collection
Milk samples were collected aseptically from 493 quarters (250 lactating goats) diagnosed with CMT >+1 and were submitted for bacteriological examination. Briefly, the udder was thoroughly washed with water and dried with a clean towel. After disinfecting the teats with 70% ethyl alcohol swabs, milk was collected. The first 3-4 streams of milk were discarded, and then, 5-10 ml of milk was collected from each teat aseptically in separate universal bottles which held a slightly horizontal position to avoid contamination from the udder [25]. Immediately, after collection, samples were cooled and transferred to the laboratory on ice and were stored at −20°C until analyzed.

Bacteriological examination of milk samples
The isolation and identification of bacterial pathogens were performed according to the procedure described by Mbindyo et al. [26]. In Brief, 0.1 ml milk sample was cultured on blood agar, nutrient agar, MacConkey agar, and Brucella agar base. The inoculated plates were incubated at 37°C aerobically for 24-48 h, while those on Brucella agar were incubated at 37°C anaerobically for 7 days. The bacterial pathogens were identified by morphology, hemolysis, gram staining, and biochemical tests such as catalase, oxidase, coagulase, reaction on sulfite, indole, and motile medium, and fermentation of sugars.

Statistical analysis
Data obtained from CMT and physical examinations were used to determine the apparent prevalence of SCM and CM (blind teat) in goats, respectively. Apparent prevalence of mastitis, clinical presentation, and isolated pathogens was determined using standard formulae, i.e., the number of positive animal/samples divided by the total of animals/samples examined.  (Table-1).
Available at www.veterinaryworld.org/Vol.12/May-2019/3.pdf The bacteriological research results enabled to identify and characterize cultures of microorganisms from milk samples of mastitis goats. Their relative prevalence rate is presented in Table-4. A total of 200 bacterial isolates were recovered from 145 (116 SCM-CMT positive milk and 29 from clinical mastitis quarters) mammary gland secretion samples at quarter level of 104 goats. S. aureus (20.0%) was the most frequent isolates from clinical and subclinical mastitic samples. The next most frequent isolates from all bacteria were E. coli (15.5%) and S. agalactiae (11.0%). Other less common isolates (≤6%) and their frequency of isolation are given in Table-4.

Discussion
Mastitis, inflammation of mammary gland, is one of the most important diseases of dairy animals worldwide [27][28][29], and it is notoriously difficult to estimate the losses associated with clinical and SCM, which arises from the costs of treatment, culling, death and decreased milk production, and constituent quality [30]. CM presents significant clinical features of inflammatory signs in udder tissues and abnormal udder secretion, whereas the only indicator of SCM is higher somatic cell count in milk without any visible abnormalities in udder tissue and milk [28]. In dairy goats, the incidence of clinical mastitis may not exceed 5%, while SCM is common and about 6 times more than clinical affections [31].   The present study showed an overall apparent prevalence of mastitis in lactating goats in three Nigerian states to be 40.4% as determined by CMT and clinical examinations of the udder. This finding is relatively higher than most of the previous findings conducted in Nigeria, where the scores range between 10% and 19.1% [30,32,33]; however, it has agreement with the results reported in other countries of the world such as 40.5% in Brazil by Oliveira et al. [34] and 43.33% in Bangladesh by Ferdous et al. [35]. Besides, the reported apparent prevalence of this study is lower compared to the higher prevalence rate 47.6%, 48.1%, and 61% which were reported in animals in urban areas of Nigeria, Egypt, and Kenya, respectively [14,26,36]. As mastitis is a complex disease involving the interactions of various factors such as differences in management and husbandry practices, geographical distribution, health status of the flock, causative agents, environmental condition (weather), nutritional status and finally the size of the study samples may be the reason for the variation in the prevalence rates between the present study and the previous ones [14,16,30].
In this study, subclinical mastitis has found to be higher than clinical mastitis. Moreover, farmers in Nigeria are not well informed about the silent cases of mastitis. A similar observation of the high dominance of SCM was recorded by several studies [35,37].
Quarter apparent prevalence of mastitis (29.4%) recorded in this study was comparable with the finding of Mdegela et al. [38] who reported quarter prevalence rate of 30% but lower than the report made by Pirzada et al. [39], Ferdous et al. [35], and Moshi et al. [40], who reported 38%, 50%, and 72.8 % in Pakistan, Bangladesh, and Tanzania, respectively.
Typically, serous mastitis is acute, and in the absence of its treatment within 10-15 h it may transform to catarrhal form of mastitis, less often to purulent-catarrhal form [21].
The apparent prevalence of blind mammary quarters (1.4%) closely agrees with the result of Bayan et al. [41] and Dinaol et al. [42] who reported certain case of quarter blindness (unfunctional or not functional quarter) in cow. A lack of screening SCM and late or not treating clinical cases could lead to acquired blindness of mammary gland. Blind mammary quarters contribute to high SCM and loss of milk production with a subsequent impact on food security [43]. The high prevalence of S. aureus followed by E. coli in this study is in accordance with other workers who found a higher prevalence of these organisms from goat's milk samples positive for mastitis [11,44]. These findings and other research works justify that S. aureus is the most important and predominant mastitis-causing pathogen globally, including Nigeria. The high prevalence of S. aureus in goats is suggested to be reinfection of mammary glands and teat lesions which transfer during milking operation, and in Nigeria, mostly, hand milking is practiced. Intramammary infections caused by S. aureus are very important from public health point of view due to its ability to produce thermostable enterotoxins and leukotoxins [45]. The higher prevalence of E. coli could be linked to the poor hygienic practices in the dairy environment. This may be attributed to the increased opportunity of infection with time and the prolonged duration of infection, especially in a herd without a mastitis control program [44].

Conclusion
Mastitis is of great economic importance to milk producers because the disease has a negative impact on several important aspects of goat and herd performance. From the present study, it was concluded that

Microorganisms
Frequency