VETERINARSKI ARHIV 69 (5), 241-250, 1999

ISSN 1331-8055 Published in Croatia

Caprine mycoplasmal mastitis in Nigeria

Godwin Oyeamechi Egwu¹*, James Agbo Ameh², Murtala Mohammed Aliyu¹, and Fatima Dogo Mohammed²

¹Department of Veterinary Medicine

²Department of Veterinary Microbiology and Parasitology,
Faculty of Veterinary Medicine, University of Maiduguri, Nigeria

* Contact address:
Dr. G. O. Egwu,
Department of Veterinary Medicine, Faculty of Veterinary Medicine, University of Maiduguri, P.M.B. 1069, Maiduguri, Nigeria.
Phone: 234 76 231900

EGWU, G. O., J. A. AMEH, M. M. ALIYU, F. D. MOHAMMED: Caprine mycoplasmal mastitis in Nigeria. Vet. arhiv 69, 241-250, 1999.

ABSTRACT

In a study to investigate the current status of inflammatory mycoplasmosis in caprine udders in Nigeria, a total of 57 and 24 milk samples were collected from udders of goats affected by mastitis and apparently normal healthy goats udders, respectively. Mycoplasma agalactiae and M. capriolum occurred significantly higher (P<0.05) in udders affected by mastitis than in normal healthy udders. Other mycoplasmas occurring in low prevalence include M. bovis and M. mycoides subsp. mycoides L.C. A study to determine the sero-prevalence of the single most prevalent mycoplasmal species, using dot enzyme immuno-assay (dot-blot) and complement fixation test (CFT) was also conducted. Seventy does affected with mastitis and 43 apparently normal healthy does selected randomly at different stages of lactation were bled. The dot-blot detected 21 (30%) and 11 (25.6%) compared with 15 (21%) and 6 (11.4%) with CFT in udders affected by mastitis and apparently normal healthy udders, respectively. The reciprocal serum CFT titres ranged from 20-160 and 20-80 udders with mastitis and apparently normal healthy udders, respectively. It is concluded that cultural (microbiological) and sero-epidemiological surveillance is necessary for effective treatment and control of the disease.

Key words: goat, udder, Mycoplasma, mastitis, Nigeria

Introduction

Mastitis is a common disease entity of dairy goats, accompanied by physical, chemical, pathological and bacteriological changes in milk and glandular tissue (BLOOD and RADOSTITS, 1989; AMEH et al., 1993). The disease is usually classified as sub-clinical, acute, subacute, chronic and gangrenous based on aetio-pathological findings and observations (HAFEEZ et al., 1987; ABU-SAMRA et al., 1988; TRIPATHI and CHALTOPADHYAY, 1993).

Predisposing factors such as poor management and hygiene, teat injuries and faulty milking machines are known to hasten the entry of infectious agents and the course of the disease (ABU-SAMRA et al., 1988; HAMMAN and EITAM, 1993; MAJIC et al., 1993).

Aetiologically, the disease is usually incriminated with multifarious agents such as bacteria, mycoplasma, yeasts and other fungi (LEPPER, 1964; DAMASSA, 1983; DAMASSA et al., 1987; ABU-SAMRA et al., 1988; RAPOPORT et al., 1993).

Goat husbandry is becoming increasingly important due to its meat and milk products (DEVENDRA and MCLEROY, 1982; HAENLEIN, 1992; EGWU et al., 1994). Moreover, goat milk provides much needed animal protein supplement, particularly in developing countries like Nigeria where goat milk consumption is gaining importance in some parts of the country (EGWU et al., 1995).

These species (goats) constitute about 26.5×106 in Nigeria (ADEMOSUN et al., 1984), the majority of which are located in the extreme northern Sahel region of the country (NGERE et al., 1984).

However, management and disease problems, particularly mastitis, constitute major constraints for effective dairy goat husbandry in Nigeria (GEFU, 1982; OJO, 1971; OJO, 1976; EGWU et al., 1994). Although the prevalence of other infectious mastitic agents from different localities has been previously reported (AMEH et al., 1993; EGWU et al., 1994), the occurrence of mycoplasmas in non-mastitic and mastitic caprine udders remains unknown in Nigeria.

There is therefore a need for comprehensive knowledge of the distribution of the main causative organisms involved in caprine mastitis in different geographical locations, in order to be to able to control and institute effective chemotherapy.

Materials and methods

Source of samples

Milk samples were collected from 24 apparently normal healthy udders (control) and 57 clinically affected udders with mastitis (both halves 24; one half 33).

The apparently normal healthy does were from 10 different sources, derived from the same flock of small- scale private backyard holders. The does sampled were located in Maiduguri, a semi-arid area of Nigeria, with a tropical climate where temperatures reach 10-15 °C from November to January, and 38-41 °C from March-May and with scanty rainfall. Most of the does sampled were semi intensively managed, fed dry legume hay/pasture and supplemented with a local concentrate called "dusa-massara" (maize offals). The breeds sampled were "local" and included Red Sokoto, Sahel and their crosses.

The number of samples collected and grouped according to the clinical state of mastitis within each age group is shown in Table 1. Does with mastitis sampled were in different stages of lactation.

Table 1. Goats with mastitis grouped according to the clinical state of the udders in various age groups

In another study, 113 randomly selected matured lactating does aged 2-4 years, 70 of them clinically affected with mastitis and 43 apparently normal healthy does, were bled for serum samples. Blood was collected in vacuitaner tubes (R) from the jugular vein, allowed to clot and sera separated and stored at -20 °C until used. Blood samples were mostly obtained from the Abattoir, University farm and small-scale backyard holders located in Maiduguri.

Sera were used to assay only for the mycoplasmal species which showed the highest prevalence following cultural (microbiological) examination of the initial 57 clinically affected samples (mentioned above).

Media, cultivation and identification of Mycoplasmas

Mycoplasmas were isolated and sub-cultured on modified Eaton's medium (Mycoplasma broth and agar) described by BOUGHTON and THORN (1993) as contained in the Central Veterinary Laboratory's (CVL) manual on Mycoplasmology. Sampled swabs were broken down into 2 ml of the mycoplasma broth medium and incubated at 5% CO₂ incubator at 39 °C for up to 3 weeks. Broth cultures which showed acidic colour change were further sub-cultured in broth and agar for further incubation as previously described by EGWU et al. (1994). The isolates were identified by agar block immuno-fluorescent specific antibody test technique as described by ROSENDAL and BLACK (1972), as well as by standard biochemical tests.

Serology

Two techniques using complement fixation test (CFT) and dot enzyme immuno-assay (dot blot) were used for the antibody assay.

Complement fixation test

This was carried out using the standard method of the office Des International Epizootics (OIE) as described by SANTINI et al. (1992). The antigen was a boiled field strain. Titres of 50% fixation at 1/20 or higher were considered positive.

Dot blotting tests

For dot blotting, mycoplasma organisms were washed twice in PBS (10 mm phosphate buffered saline, pH 7.2) and re-suspended in 20 ul of PBS. About 1-2 ul of each 4 serial doubling dilution from 1:10 were placed on nitrocellulose strips. When dry, the membrane was treated with 5% skim milk solution in PBST (PBS, pH 7.2) 0.5% Tween 20 for 1 hr at 37 ^oC and washed 5 times in PBST. Test sera, diluted 1:400 and 1:800, were incubated with the strips for 1 hr at 37 °C, washed 5 times in PBS, and incubated in anti-goat 1 gG alkaline posphate congugate (1:5000) for 1 hr at 37 °C. Five further washed in PBS was followed by the detection of adherent antibodies with 5 bromo-chloro indolyl phosphate (BCIP)/nitroblue tetrazolium (NBT) substrate (Dynatech, U.K.). Interpretation of the test was subjective and was based on the intensity of staining compared to known positive and negative sera.

Statistics

The level of significance between the occurrence of M. agalactiae and M. capricolum in udders with mastitis and udders without mastitis (control) was determined using the c² test and all values where P<0.05 were considered as significant.

Results

Four and 5 samples were obtained from non mastitic and mastitic udder, respectively, from goats aged between 9 months and 1 year, while most samples were obtained from goats aged between 1 and 3 years either in the non mastitic (20) and mastitic (52) udders. More acute (29) and chronic (15) udders were more commonly observed within this age group (Table 1).

The prevalence of Mycoplasma and Acholeplasma species in each of the 24 non-mastitic and 57 mastitic udders is shown in Table 2. A total of 5 Mycoplasma and Acholeplasma species were isolated from the mastitic cases, while only two species of Mycoplasma and one of Acholeplasma were recovered from non-mastitic udders.

Table 2. Prevalence of Mycoplasma species in each of 24 non-mastitic and 57 mastitic caprine udders from Nigeria

Figures in brackets are the percentage prevalence among the Mycoplasma species determined in each of the total 24 unaffected and 57 affected caprine udders
Values denote by different superscripts for a given parameter were significantly different (P<0.05)

Mycoplasma agalactiae and M. capricolum occurred significantly more frequntly (P<0.05) in udders affected with mastitis than in apparently normal healthy goats and udders. Two non-host specific Mycoplasma or Acholeplasma (M. putrefaciens and A. laidlawi) were also recovered either in the apparently normal healthy udders (control) and udders with mastitis. These species occurred with other Mycoplasma species, such as M. bovis or M. mycoides subsp mycoides LC, but in low prevalence.

The serological prevalence of the most frequent occurrence of mycoplasma (M. agalactiae) in 70 mastitic and 43 apparently normal health randomly selected does is shown in Table 3. Dot blot ELISA was more sensitive in detecting serological presence of this organism (M. agalactiae) in 21 (30%) and 11 (25.6%) compared with 15 (21%) and 6 (11.4%) with the CFT in mastitic and non-mastitic udders, respectively.

Table 3. Number of positive serum samples to complement fixation test (CFT) and dot enzyme immuno-assay (dot-blo) to Mycoplasma agalactiae in 70 sera from unaffected does from Nigeria

The reciprocal CFT titres in either the mastitic and non-mastitis udder ranged from 20-160 and 20-80, respectively.

The clinical description of the various clinical states of caprine mastitis has been described by EGWU et al. (1994).

Discussion

Prior to this present investigation, no microbiological examination has been performed on the prevalence of mycoplasmas in mastitic and non-mastitic caprine udders does in Nigeria. Therefore, it is likely that some of the previously diagnosed cases of caprine mastitis may have been associated with mycoplasmas, either single or in combination with other infectious agents incriminated with this disease. It seems obvious that mis-diagnosis constitutes persistent problem in the accurate aetiological diagnoses of caprine mastitis in Nigeria. This is so because cultural (microbiological) examination of these organisms (mycoplasmas) is seldom difficult to carry out in most diagnostic laboratories of the developing countries.

All does in various stages of lactation used in this study were managed semi-intensively. This management system usually predisposes goats to mastitis due to injuries (abrasions, perforations and ulcerations) on teats (ABU-SAMRA et al., 1988). Some of these injuries occurred more often in subacute/acute cases, and it is likely that this may have facilitated the entry of some of the mycoplasmal organism, similar to the observations of DEVENDRA and MCLEROY (1982).

During this present investigation, fewer samples were collected within the age group of 9 months to 1 year. This can be accounted for by the fact that does within this age group are not routinely exposed to causal agents of mastitis, since most of the does are near to or in their first lactation. Milk production is known to increase with milking and subsequent lactation (s), which may probably explain why does within the 1-3 year age group were more commonly affected in this present study. Moreover, subsequent kidding has been shown to increase the chances of infection (GROSS et al., 1978). Hence, does more than 4 years old were either becoming unproductive or were culled for market to meet family financial demands.

More acute (29) and chronic (15) cases were observed in does aged 1-3 years than in sub-acute (5) and gangrenous (3), as similarly reported by BLOOD et al. (1990). This may be associated with the fact that following kidding the udders become fully or partially engorged, which then induces patency in the teat canal thereby facilitating entry of susceptible and infectious organisms. On the other hand, following post-weaning, when the udder is becoming empty, progression of acute to chronic disease can occur if there is no effective chemotherapy to eliminate causative agents, or vice-versa if infectious agents persist following subsequent kidding.

The predominance of Mycoplasma agalactiae and M. capriolum in caprine udders in this present study corroborates the observations of SMITH and ROGUINSKY (1977) and BLOOD et al. (1990). These authors have reported and implicated these species as being more commonly encountered in acute and chronic states. Therefore, their occurrence in this present investigation does indicate a possible causal role or carrier status in mastitic and non-mastitic udders.

Although most mycoplasmas are host specific (JONES, 1983), the occurrence of M. putrefaciens and A. laidlawii could be regarded as commensals or agents of unknown pathogenicity. Moreover, the occurrence of  M. bovis, which is a known bovine pathogenic mycoplasma (JASPER, 1982; BERTHOLD et al., 1992), could have resulted from cross infection as a result of mixed herding (grazing) of sheep, goat and cattle. Mycoplasma mycoides subsp mycoides L.C. has been incriminated and isolated from sheep and goats with septiceamia, arthritis and mastitis in various clinical states of disease (MACHARIA et al., 1995). It is likely that the occurrence of this species in the udder may have resulted from any of the above clinical disease states.

Serological evidence to corroborate the prevalence of the single most frequently occurring mycoplasmal species (M. agalactiae) with cultural (microbiological) studies using dot immuno-enzyme assay and complement fixation, showed that the former was a sensitive immuno-diagnostic technique. The results obtained correspond with the cultural (microbiological) examination of mycoplasmas. It further shows that the total increased prevalence of 32 in mastitic and non-mastitic does may suggest evidence of previous or recent infection. This is further supported by the reciprocal CFT antibody titre, which ranged from 20-160 in mastitic compared with 20-80 in non-mastitic cases.

The present study, in conjunction with previous reports (AMEH et al., 1994; EGWU et al., 1994) has fully established the prevalence of mastitic infection in Nigeria. The authors have further highlighted the multifactorial and diverse involvement of infectious agents in clinical mastitis.

There is need to put emphasis on mycoplasmal mastitis, particularly in Nigeria, where diagnosis and treatment are usually based on clinical observation (s) rather than on cultural (microbiological) examination of the likely causative organism (s).

In conclusion, this study reports for the first time the most prevalent mycoplasmal species, as well as serological evidence of the most commonly occurring species.

There is also a need for an effective microbiological or serological examination for accurate diagnosis and epidemiological surveillance in order to enhance effective treatment and control of this disease (mastitis).

Acknowledgements
The senior author is grateful to Rockefeller Foundations U.S.A. for a biotechnology career Fellowship tenable at Central Vet. Laboratory, Weybridge England, where the immunological and some of the cultural (microbiological) examinations were carried out. The expertise of Robin Nicholas and the kind assistance provided by Kathy Clarke Samantha Baker (mycoplasmology section) during analysis of the samples is duly appreciated.

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Received: 18 February 1998
Accepted: 6 September 1999

EGWU, G.O., J. A. AMEH, M. M. ALIYU, F. D. MOHAMMED: Mikoplazmatska upala vimena koza u Nigeriji. Vet. arhiv 69, 241-250, 1999.

SAZETAK

U istrazivanju stanja upalnih mikolplazmoza kozjih vimena u Nigeriji pregledani su uzorci mlijeka iz ukupno 57 oboljelih od upale vimena i iz 24 zdravih kozjih vimena. U vimenima s upalom vimena pronadeno je znacajno više (P<0,05) Mycoplasma agalacticae i M. capriolum nego u normalnim zdravim vimenima. Druge mikoplazme koje su se rijede pojavljivale bile su M. bovis i M. mycoides subs. mycoides L.C. Provedeno je i istrazivanje da se utvrdi serološka prevalencija pojedine najucestalije vrste mikoplazme, uporabom tockastog encimskog imunološkog testa (Dot-blot) i testa komplementarne fiksacije (CFT). Uzeta je krv od 70 koza s upalom vimena i od 43 zdrave i nasumicno odabrane koze u razlicitim stadijima laktacije. Metoda Dot-blot je pokazala pozitivnu reakciju u 21 (30,0%) oboljelih vimena i 11 (25,6%) u zdravih vimena, a CFT u 15 (21,0%) oboljelih i 6 (11,4) zdravih vimena. Reciprocni serumski CFT titrovi bili su u rasponu od 20 do 160 u oboljelih i 20 do 80 u zdravih kozjih vimena. Zakljuceno je da je potrebno provoditi mikrobiološka (uzgoj kultura) i seroepidemiološka istrazivanja za ucinkovito lijecenje i kontrolu bolesti.

Kljucne rijeci: koza, vime, Mycoplasma, upala vimena, mastitis, Nigerija