Aerobic Bacteria Pathogens Associated with Caprine Mastitis in Nsukka Area of Enugu State

This study ascertained the aerobic bacteria associated with cases of clinical and subclinical caprine mastitis. A total of 58 lactating West African dwarf does were used for this investigation. These lactating does had signs of clinical and sub-clinical mastitis. These samples were collected from Nsukka town, Nsukka abattoir, Orba Market, Obukpa Market and Obolllo Afor market, all in Enugu State, Nigeria. Clinical mastitis was detected by gross signs of udder infection by physical examination of abnormal milk, whereas subclinical mastitis was recognized using California Mastitis test. This study showed that 39 (67.24%) of 58 goats were positive for mastitis on California Mastitis Test. Clinical and subclinical cases of caprine mastitis, Staphylococcus aureus was the predominant pathogen 32 (55.17%). 14 (24.13%) of the isolates were positive for Staphylococcus epidermidis, 6 (10.34%) for Streptoococcus agalactiae, 4 (6.89%) E.coli and 2 (3.44%) for Klebsiella pneumoniae. This work identified the bacterial species linked with caprine mastitis in Nsukka area of Enugu State.


Introduction
In Nigeria, one of the major limiting factors to the successful rearing of goats and sheep is disease, mastitis is a major one (Ugochukwu, 2008). The importance of goats as a source of meat and dairy products has been well discussed and documented by Haenlein (2004). Nigeria have an estimated goat population of 34.5 million (Lawal-Adebowale, 2012) but very little of this large population, is used for diary purposes (Ugochukwu, 1983b). For this reason, little attention has been directed to study the incidence and proffer control solution for caprine mastitis.
Mastitis, an inflammatory reaction of the udder tissue to bacterial, chemical, thermal or mechanical injury is characterised majorly by physical, chemical and bacteriological changes in milk with attending necrotic, pathological and glandular structural changes in defined areas of the udder (Togun et al., 2003). Mastitis is one of the major diseases in veterinary medicine. The global economic losses due to mastitis is about $35 billion annually. In addition to the huge direct and indirect economic losses, the presence of certain pathogens in the milk is a major threat to public health (Bedolla and Castañeda, 2003;Bilal et al., 2004;Wolter et al. 2004;Ali et al., 2010). Mastitis is a complex disease with multiple predisposing factors and various causative agents (Harmon et al., 1990;Radostits et al., 2000;Tollersud et al., 2000;Bedolla, and Castañeda 2003;Bergioner et al., 2003). It is a disease complex which is dependent on several factors such as aetiologic agents, management practices such as the use of milking machine, susceptibility to the disease and efficiency of the host defence mechanisms (Ugochukwu, 1983b). Clinical mastitis may be easy to detect but animals suffering from subclinical mastitis are very difficult to diagnose since there is a lack of reliable  (Leitner et al., 2004). Early diagnosis of subclinical mastitis is vital Because changes in udder tissue take place much earlier than they become apparent (Contreras, 2007). However, the diagnosis of subclinical mastitis in goats is not easy but direct bacteriological assay could be used (Maisi and Riipinen, 1988;Maisi, 1990;Fthenakis, 1995;Gonzalez-Rodriguez and Carmenes, 1996;Sanchez et al., 2004).
Clinically, affected glands frequently suffer partial or complete damage and do not resume normal function (Fthenakis and Jones, 1990;Mørk et al., 2007). Most of the cases of mastitis studies show serious bacteria aetiological involvement. Many bacterial strains has been implicated in the aetiology of caprine mastitis, some frequently isolated organisms includes: Staphylococcus spp. Streptococcus. spp, E.coli, Corynebacterium spp. and Nocardia spp (Ugochukwu, 1983a;Ameh and Tari, 2000;Ajuwape et al ., 2005Mørk et al.,2007. Some other bacterial species incriminated include Pseudomonas aeruginosa, Mannheimia haemolytica, Klebsiella spp, Pasteurella spp, Listeria monocytogenes and Fungi (Adetunji and Olaoye, 2012;Hawari et al., 2014). Control measures for mastitis are important in order to reduce economic losses and decrease the risk of diseases, being transmitted to humans via food (Cortimiglia et al., 2015).
There has been little knowledge of the aetiology of this disease. Goat and sheep industries is gaining grounds in some part of Nigeria, coupled with the dearth of information in available scientific literature on caprine mastitis in Nsukka area and the Southeastern part of Nigeria, necessitated this bacteriological investigation.

Study area
The study areas are Nsukka and Obukpa market in Nsukka Local Government Area, Orba and Obollo Afor market in Udenu Local Government Area, Enugu State, Nigeria. Nsukka is situated at latitude 6°51'24"N and longitude 7° 23'45"E, with a total Landmass of 17.52 sq. miles (45.38km 2 ), an annual mean humidity of 13% and an elevation of 1,810ft (550m) above sea level. The geographical coordinates for Orba is 6⁰51'0''N, 7⁰27'0''E, while Obollo is located at 6⁰55'N 7⁰31'E.

Study animals
Animals used for this investigation were 58 lactating West African dwarf does with obvious clinical signs of clinical and sub-clinical mastitis.

Sample collection
Samples were collected from clinical and subclinical cases of mastitis. The milk samples were collected under complete aseptic conditions.

Diagnosis of Clinical and sub-clinical mastitis
Clinical mastitis was detected by gross signs of udder infection during physical examination of abnormal milk, while subclinical mastitis was recognized using California Mastitis test (CMT) following standard procedures described by Assefa, et al ., (2006). Milk samples of the goats with clinical and subclinical mastitis were subjected for isolation and identification of bacterial pathogens.

Isolation and identification of bacterial agents
Samples were kept in the refrigerator at 4°C within a maximum of 12h after sampling before examination. The samples were centrifuged at 1500 rpm for 30min as recommended by Yousof -Beighi et al. (2005). The sediments were cultured in Blood agar, MacConkey agar and Nutrient agar plates. The inoculated plates were incubated under aerobe conditions at 37°C and colonial growth was checked after 24 h. The colonial morphology and type of haemolysis were recorded. After Gram staining, the suspected pure colonies of gram positive were also identified by culturing in Mannitol salt agar, Catalase Test and Tube Coagulase Test using standard procedures as described by Chessbrough (2000). The Gram negative rods were subjected to Giemsa staining, indole, sugar utilization and urease tests as well as reaction on eosin methylene blue agar and Triple sugar iron agar (TSI agar) according to standard procedures described by Chessbrough (2000). Identification of the bacterial agents from the pure culture were based on colony characteristics, gram staining reaction, hemolysis pattern and biochemical test described by Chessbrough (2000).

Bacteriological findings
The distribution of aerobic bacteria isolated from secretions from the glands with clinical and subclinical mastitis is shown in Figure 1.
Staphylococcus aureus was the predominant pathogen and was 32 (55.17%) of the samples from the mastitic glands. 14 (24.13%) of the isolates were Staphylococcus epidermidis, 6 (10.34%) of the isolates were Streptoococcus agalactiae, 4 (6.89%) of the isolates were positive for E. coli and 2 (3.44%) of the isolates were positive for Klebsiella pneumoniae as shown in Figure 2.

Bacteriological findings
The distribution of aerobic bacteria isolated from secretions from the glands with clinical and subclinical mastitis is shown in Figure 3. Staphylococcus aureus was the predominant pathogen and was 32 (55.17%) of the samples from the mastitic glands. 14 (24.13%) of the isolates were Staphylococcus epidermidis, 6 (10.34%) of the isolates were Streptoococcus agalactiae, 4 (6.89%) of the isolates were positive for E. coli and 2 (3.44%) of the isolates were positive for Klebsiella pneumoniae as shown in Figure 2.   Caprine mastitis is caused by bacteria of primary or secondary involvements. The udder could also be exposed to trauma from hard objects like broken bottles and rusty metals in their scavenging environment, which make the goats susceptible to mastitis -causing organisms Ugochukwu et al.

Bio-Research
Published January/December 2017 ISSN 1596-7409 (Togun et al., 2003). Raw goat's milk can be a potential source of antibiotic resistant pathogens on animal, human, and environment. Microorganisms which contaminate raw milk may originate from the farm environment or goats which include aetiological agents responsible for clinical and subclinical mastitis (Virdis et al., 2010). Out of the 58 isolates, Staphylococcus aureus had the highest isolation occurrence of 32 (55.17%). 9 (28.12%) out of the 32 Staphylococcus aureus isolates were Coagulase negative Staphylococcus aureus. Coagulasenegative staphylococci isolated in this study, although not a major pathogen of clinical mastitis in goats, has been shown to persist throughout the lactation and dry periods, irritating the gland and causing a decrease in production and even clinical mastitis (Ndegwa et al., 2001).

In this study the bacterial isolates include
Staphylococcus aureus has been incriminated in much suppurative conditions in man and animals (Greenwood et al., 2002;Kitara et al., 2011). Coagulase positive Staphylococcus aureus is known to be involved in mastitis in most species of domestic animals including goats, pigs, cattle and sheep (Radostits et al., 2000). It has also been isolated with the highest frequency in other works as reported by Mørk et al., (2007), Ali et al., (2010 and Islam et al., (2011). The occurrence of a high number of coagulase positive Staphylococcus aureus suggest its involvement in the causation of caprine mastitis. Staphylococcus epidermidis isolated in this study is widely known to be harmless and the precise role of these bacteria in the pathogenesis of mastitis is not understood but has been associated with subclinical mastitis. It has been isolated by Contreras et al., (2003) and Razi et al., (2012).
Escherichia coli is a normal microflora of the gut. It is usually present in the faecal materials. Contamination of the udder by faecal materials could have led to the presence of the organism in the milk. Isolation of E. coli in this study agrees with the findings of Islam et al., (2011), Hawari et al., (2014 and Mbindyo et al. (2014).
Streptococcus agalactiae, a notorious agent for mastitis (Kateeke et al., 2013). Castañeda et al., (2013) and Hawari et al. (2014), reported isolation and identification of this organism from clinical cases of mastitis in their works on mastititis in caprines, bovines and ovines, in Northern Nigeria, Mexico and Al-Balqa province in Jordan, respectively.
The differences in the Breed distribution of Mastitis, in which there was more cases in WAD goats compared to Kano Brown goat is due to the fact that WAD goats are the predominantly reared goat breed in Southeastern Nigeria.
In the present circumstances, goats and sheep industry is gaining considerable attention since 1983 as reported by Ugochukwu, 1983b. It is important as part of control measures to encourage indigenous farmers to report cases of mastitis to the nearest veterinary clinics.
In conclusion, this work identified the bacterial species involved in caprine mastitis in Nsukka area of Enugu State. It is believed that this investigation will stimulate research on other pathogens associated with caprine mastitis Bedolla, C.C. and Castañeda, V.H. (2003).