Trop Anim Health Prod DOI 10.1007/s11250-010-9717-9
ORIGINAL RESEARCH
Seroprevalence of and risk factors for Mycoplasma mycoides subspecies capri infection in small ruminants in Northern Jordan Waleed Al-Momani & Mahmoud N. Abo-Shehada & Robin A. J. Nicholas
Accepted: 8 October 2010 # Springer Science+Business Media B.V. 2010
Abstract During the period from January 2002 to December 2003, serum samples were collected from 104 small ruminant flocks consisting of 18 sheep flocks, 27 goat flocks, and 59 mixed flocks containing both sheep and goats in northern Jordan. Only female sheep and goats were sampled. At least five females aged over 2 years per flock per species were sampled and examined for Mycoplasma mycoides subspecies capri using the latex agglutination test. To increase the chances of detecting positive flocks, sick or older ewes were sampled. Specific information was obtained using a questionnaire to identify potential risk factors for M. mycoides subsp. capri seropositivity in small ruminants. The true flock-level seroprevalences of M. mycoides subsp. capri were 34%, 32%, and 38% in small ruminants (sheep and goats), sheep, and goats, respectively. Differences between flock-level seroprevalences in sheep and goats were not significant (p=0.7). Multivariable logistic regression analysis of 21 production and health management practices showed four to be associated with M.
W. Al-Momani (*) Department of Allied Medical Sciences, Al-Balqa’ Applied University, P.O. Box 19117, Al-Salt, Jordan e-mail:
[email protected] M. N. Abo-Shehada Department of Basic Medical Veterinary Sciences, Faculty of Veterinary Medicine, Jordan University of Science and Technology, P.O. Box 3030, Irbid 22110, Jordan R. A. J. Nicholas Department of Bacterial Diseases, Mycoplasma Group, Veterinary Laboratories Agency-Weybridge, New Haw, Addlestone, Surrey KT15 3 NB, UK
mycoides subsp. capri seropositivity including flocks which were grazed and fed concentrate supplement (OR= 4.6), improper cleaning of milking utensils (OR=4.7), buying new animals to replace culled ones (OR=0.3), and treating against helminths when clinical signs of helminth infections appear (OR=0.4). Keywords Mycoplasma mycoides subspecies capri . Small ruminant . Sheep . Goat . Seroprevalence . Risk factors . Jordan
Introduction Mycoplasma mycoides subspecies capri is one of four mycoplasmas designated by the World Animal Health Organization (OIE) as a cause of contagious agalactia, together with Mycoplasma agalactiae, Mycoplasma capricolum subspecies capricolum, and Mycoplasma putrefaciens (Nicholas and Loria 2008). The relation between M. mycoides subsp. mycoides LC and M. mycoides subsp. capri is contentious, with many workers considering the two to be identical particularly on the basis of 16S rRNA sequencing which reveals a 99.9% similarity (Pettersson et al. 1996) and 99% similarity by sequencing of a putative membrane protein gene (Thiaucourt et al. 2000). A recent study using different molecular, biochemical, and serological techniques supported the reclassification of the M. mycoides subsp. mycoides LC and M. mycoides subsp. capri strains into the single subspecies, M. mycoides subsp. capri (Shahram et al. 2010). M. mycoides subsp. capri has been isolated from goats and occasionally from sheep with balanoposthitis and vulvovaginitis (Trichard et al. 1993). In Europe, M. mycoides subsp. capri is a major cause of contagious
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agalactia in goats (Loria et al. 2003). In western France more than 50% of the isolated mycoplasmas from bulk milk tank were M. mycoides subsp. capri (Mercier et al. 2001). In mainland Spain, M. mycoides subsp. capri accounts for 7% of the isolates from the affected goat flocks (Gil et al. 1999). Also, the most frequently occurring caprine Mycoplasma infections in the USA were reported to be due to M. mycoides subsp. capri (Smith and Sherman 1994). M. mycoides subsp. capri infection causes economic losses through decreased production, mortality, diagnosis, and treatment costs; the losses can be high in severe outbreaks particularly in intensive commercial goat diary operations (Nicholas 1998). In extreme cases, septicemic goats may die without clinical signs (Di Provvido et al. 2009). Clinical signs associated with M. mycoides subsp. capri infections in goats include pleuropneumonia, arthritis, mastitis, keratoconjunctivitis, and septicemia (Nicholas and Loria 2008). In kids, experimental infections with M. mycoides subsp. capri result in fever, acute pain, swollen joints, and dyspnea, often with death as a result of septicemia (Nicholas et al. 2008). Three small ruminant mycoplasmas, M. mycoides subsp. capri, M. capricolum subsp. capricolum, M. putrefaciens were isolated from Jordan (Al-Momani et al. 2006). The seroprevalence of M. mycoides subsp. capri has not been studied in Jordan. This study investigates the seroprevalence of M. mycoides subsp. capri among sheep and goat flocks in northern Jordan along with some risk factors for M. mycoides subsp. capri seropositivity.
Materials and methods Study area and animals The study area included the five northern governorates of Jordan: Irbid, Mafraq, Jerash, Ajlon, and Zarqa. The main small ruminant farming activities occur within three climatic zones: the warm desert, cool temperate rainy, and the cool steppe. In Jordan, small ruminants are the most abundant domestic animals in Jordan; 74% are sheep, 23% goats, and 2% cattle (Anonymous 2001). Sheep and goats are usually kept together mainly under free-range roaming transhumance husbandry with a small number of flocks adapting semi-intensive husbandry methods. Flocks are grazed during spring to early summer and fed on supplements of fodder and concentrates for the rest of the year. Some farmers give additional concentrates during the grazing season. The main sources of drinking water for sheep and goats are springs and piped water. The lambing season lasts from November until May. Animals are housed together in one barn. Some farmers have a special barn for
newborns, where newborn lambs may be kept with or without their dams. The routinely recommended control measure for infectious disease is vaccination, and no vaccine is used against mycoplasmosis. Sample size determination and sampling The prevalence of M. mycoides subsp. capri infections in northern Jordan was not reported previously, but assumed to be 50%. The total number of sheep and goat flocks in northern Jordan is estimated at 10,000 (Anonymous 2001). According to Thrusfield (1995), the appropriate number of flocks to be examined is 62 because 95% level of confidence and 5% absolute precision were required. The study had at least 80% power at the 5% significance level to detect an odds ratio (OR) ≥2 for risk factors present in 50% of controls and an OR≥3 for those present in 20% of controls. The main communal grazing areas were sampled including at least one flock per grazing area. Representative flock samples were selected according to the estimated density in each area. The flocks were numbered in each grazing area, and then a number was drawn to be interviewed and sampled. On seven occasions, farmers declined sampling for different reasons: they did not like blood drawn from their animals; the owner was not present to give the permission, the absence of adult male family members, in which case, another number was drawn. A number of flocks (39) from an unshared grazing area (either private or common grazing area with no access to other farmers) were included. Thus, 104 flocks (18 sheep, 27 goat, and 59 mixed) having 12,093 sheep and 4,225 goats were randomly selected. The maximum number of sheep per flock is 620 and the minimum is 20 (quartiles are Q1= 40, Q2=90, and Q3=200). The maximum number of goats per flock is 300, whereas the minimum is eight (quartiles are Q1=15, Q2=25, and Q3=57). Due to limited funds, the selection of individual sheep/ goats for testing included the animals most likely to be infected in the flock (older or in poorer condition). According to Cannon and Roe (1982), the sample size required to be 95% confident that disease is present in the flock at a prevalence of 50%, is five animals. Thus, five to ten female animals more than 2 years of age per flock per species were sampled. All single-specie flocks (18 sheep and 27 goat) and 20 mixed flocks were sampled from both species, and 39 mixed species flocks were sampled from either sheep (n=24) or goat (n=15) depending on the main species in the flock. During the period from January 2002 to December 2003, each farm/flock was visited once for blood and data collection. Five and ten samples were collected from 55 and nine sheep flocks respectively. Five, six, nine, and ten
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samples were collected from 59, one, one, and one goat flocks, respectively. Five-milliliter blood samples were obtained from the jugular vein of each ewe (n=678) in a vacuum tube without anticoagulant. Serum samples were separated and stored in aliquots at −20°C until used. Data collection A questionnaire was designed to collect information in a personal interview with the farmer using closed questions. This information covered 12 production and ten health management practices. The questionnaire was filled by direct interview with the farmers and conducted by a veterinarian who spoke in the same dialect of Arabic as the farmers, to reduce the inaccuracies caused by miscommunication. Latex agglutination test The latex agglutination test used in this study was adapted from the test used for M. capricolum subsp. capripneumoniae (Rurangirwa et al. 1987). The adapted latex agglutination test was developed and optimized at the Veterinary Laboratories Agency (Weybridge), UK. Briefly, the capsular polysaccharide antigens from a 72-h culture of strain F30 of M. mycoides subsp. capri were purified using methods based on that of March et al. (2002). The test was performed by mixing 10 μl of latex beads bounded to polysaccharide antigen extracted from fresh M. mycoides subsp. capri (F30) culture with 10 μl of serum on a glass slide and shaken for up to 2 min. Samples causing visible agglutination against a dark background were recorded as positive (Rurangirwa et al. 1987). Binding and test format were optimized, and specificity and sensitivity of the reagents was then calculated using known positive and negative sera. Tests were scored at +++ (agglutination within 1 min), ++ (agglutination between 1 and 2 min), + (agglutination between 2 and 3 min), and –ve (no agglutination within 3 min) This test's sensitivity and specificity are 90% and 95%, respectively. Positive and negative control samples were tested in this sheep and goat latex agglutination system. A flock was considered seropositive when one or more samples were positive. Statistical methods Seroprevalences of M. mycoides subsp. capri infections were calculated and analyzed at individual and flock levels for sheep and goats. The test sensitivity and specificity were used to calculate the individual-level true prevalence according to Rogan and Gladen (1978). For calculating the flock-level true seroprevalence, the test sensitivity and specificity were adjusted from individual-level to flock-
level according to Jordan (1995) using the Herdacc program: for a median flock size of 90 animals, cut-off point of one animal positive indicating a positive flock, five to ten animals sampled per flock, and sampling without replacement. Then, flock-level sensitivity and specificity were used to calculate the flock-level true seroprevalence according to Rogan and Gladen (1978). The 95% confidence intervals were calculated for individual- and flocklevel true seroprevalences. Chi-square test was employed to test the significance between seroprevalences. Statistical analyses were performed by using SPSS software version 11 (SPSS Inc., Chicago, IL, USA) and Epi-Info (CDC, Atlanta, GA, USA). P value of
