Detecting Mollicutes by PCR in goats in southwestern Bahia, Brazil

Abstract

Brazil has a herd of over 9 million goats, and the northeast of Brazil is home to over 93% of this herd. Caprine mycoplasmosis are widely disseminated worldwide, being highly contagious with high rates of morbidity and mortality, causing considerable economic loss to goat herders. In addition, there has been a lack of research using molecular testing to monitor the health and detect Mollicutes in this herd in Brazil. Therefore, the aim of this study is to associate animal management with the presence of the caprine origin Mollicutes in goats, in the southwest region of the state of Bahia, Brazil. A cross-sectional study was conducted on twelve farms, and statistical analyses were performed to identify associations between the presence of Mollicutes and the management of goats. Molecular testing identified Mollicutes class, Mycoplasma agalactiae (Ma) and M. conjunctivae (Mc), in the samples analyzed. Statistical associations were observed between animals from intensive livestock facilities and the presence of Mollicutes in nasal samples and dairy ranch animals and the presence of Mollicutes in ocular samples and animals from extensive ranching sites and positive results of Mollicutes in genital samples. We conclude that mycoplasmas are present in goat herds in the southwestern region of Bahia, which supports the need for more focused studies of mycoplasmas throughout the country. Our research also demonstrated the presence of two important opportunistic bacteria, Mc and Ma, and, to the best of our knowledge, this is the first time that M. conjunctivae was detected in Brazilian goats by molecular testing.

Supplementary Information

The online version contains supplementary material available at 10.1007/s42770-021-00517-z.

Introduction

The Brazilian Institute of Geography and Statistics (IBGE) reports 9.6 million goats in Brazil. The northeastern region of Brazil is home to more than 90% of this total, particularly the states of Bahia, Pernambuco, Piauí and Ceará [1]. Goat herding in this area is of local economic, social, and cultural importance. Mycoplasma spp. are well-known disease-causing bacteria in ruminants. Mycoplasma agalactiae causes caprine contagious agalactiae (CA); M. conjunctivae is the main etiological agent in infectious keratoconjunctivitis in goats (IKC); and M. mycoides subsp. capri (Mmc), M. capricolum subsp. capricolum (Mcc), and M. capricolum subsp. capripneumoniae (Mccp) cause pleuropneumonia, mastitis, polyarthritis, and conjunctivitis, and they are the most important species for small ruminants [2].

Caprine mycoplasmosis has important socioeconomic consequences worldwide and particularly in regions that depend on goat herding as a source of income and food, as in Northeastern Brazil. The participation of animals in crowded markets or exhibitions [3] and scarce sanitary management diminishes the immunity of the herd, lowering productivity and raising the mortality related to the mycoplasmosis [4]. There has been a lack of consensus and standardization of protocols for determining infection, ranging from visually detecting signs in the animals, culturing bacteria, and using more definitive molecular techniques [5], and this has resulted in a lack of consistent and effective local mycoplasma detection [6]. This alarming scenario regarding sanitary conditions of the herd remains a challenge for researchers. Therefore, the aim of the present study is to use state-of-the-art molecular techniques to associate the sanitary conditions of goat herds and clinical parameters with the presence of the caprine origin Mollicutes in goats, in the southwest region of the state of Bahia (Brazilian Northeastern), Brazil.

Materials and methods

A cross-sectional study was conducted between June 2010 and September 2011 on twelve ranches located in Southwest Bahia state, Brazil. The laboratory and statistical procedures were conducted between January 2015 and June 2017 at the facilities of the Institute of Biomedical Science and Department of Preventive Veterinary Medicine and Animal Health of the Faculty of Veterinary Medicine and Animal Science of the University of São Paulo, Brazil. All procedures were carried out in agreement with the guidelines of the Ethical Principles in Animal Research adopted by the Ethics Committee in the Use of Animals of the Faculty of Veterinary Medicine and Animal Science of the University of São Paulo (protocol no. 8445300117).

Sample selection

The goat ranches were selected by an extension project of the State University of Southwest Bahia (UESB), in the cities of Vitória da Conquista, Itapetinga, Brumado, Anagé, and Bom Jesus da Serra. Animals were randomly selected, and nasal, genital, and ocular conjunctiva swab samples were collected from 104 females and 28 males, totaling 396 samples. Samples were added to 4 mL of SP 4 transport medium and stored at -20 °C until further analysis.

Epidemiological questionnaire

Questionnaires with close-ended questions were applied to investigate the zootechnical and sanitary conditions of the herds. Questions were related to breed (beef, dairy cattle), production system (intensive or extensive), type of farming (commercial or subsistence), type of milking (manual or mechanical), segregation of age groups, the use of artificial insemination technique, natural mating, cases of abortion, and presence of nasal discharge. Regarding sanitary management, questions focused on the use of disinfectant in the milking environment, the hygiene of the udders before and after milking, and the use of gloves in manual milking.

Physical examination was performed to evaluate vital parameters (heart rate, respiratory rate and temperature), lymph nodes, ocular and genital mucous articulations, pulmonary sound, and mammary glands by direct and indirect inspection through California Mastitis Test. All variables were dichotomized to facilitate tabulation and data analysis.

The complete data of the epidemiological questionnaire are provided on the supplementary data.

Molecular detection

The detection of Mollicutes was performed using conventional PCR described by Van Kuppeveld et al. [7]. Positive samples for Mollicutes were submitted to specific conventional PCR to detect M. agalactiae [8], using adjusted thermal profiles an initial cycle at 94 °C for 5 minutes, 35 cycles at 94 °C for 45 seconds, 58 °C for 60 seconds and 72 °C for 120 seconds with a final cycle at 72 °C for 10 minutes, M. conjunctivae [9], M. capricolum subsp. capricolum [10] and M. mycoides subsp. capri [11] M. c. capripneumoniae, however, were detected using Real-Time PCR [12] (Table 1).

Table 1.

Description of the PCR parameters

	Stage 1		Stage 2			Stage 3	Reference
Mollicutes	94 °C, 5 min		34 cycles			72 °C, 10 min	Van Kuppeveld et al. (1992) [7]
			94 °C, 30 seg	57.5 °C, 30 seg	72 °C, 30 seg
Ma	94 °C, 5 min		35 cycles			72 °C, 10 min	Chávez-González et al. (1995) [8]
			94 °C, 45 seg	58 °C, 60 seg	72 °C, 120 seg
Mc	50 °C, 2 min	95 °C, 10 min	40 cycles			97 °C, 15 min	Giacometti et al. (1999) [9]
			95 °C, 15 seg	58 °C, 60 seg	60 °C, 60 seg
Mcc	94 °C, 5 min		35 cycles			72 °C, 7 min	Monnerat et al. (1999) [10]
			94 °C, 30 seg	51 °C, 60 seg	72 °C, 60 seg
Mmc	94 °C, 5 min		34 cycles			72 °C, 10 min	Monnerat et al. (1999) [11]
			94 °C, 30 seg	57.5 °C, 30 seg	72 °C, 30 seg
Mccp	95 °C, 10 min		40 cycles				Fitzmaurice et al. (2008) [12]
			95 °C, 30 seg	47 °C, 60 seg	72 °C, 60 seg	55–95 °C*

*Continuous fluorescence measurement every 0.25 °C to record melting temperature

Positive controls M. agalactiae (RJ), M. conjunctivae (2002), and M. capricolum subsp. capricolum (1972) were provided by the laboratory of Mycoplasmas of ICB/USP under the direction of Prof. Dr. Jorge Timenetsky, and the strain M. mycoides subsp. capri was provided by Prof. Dr. Caio Cordova from the Regional University of Blumenau/SC. They were cultured in SP4 media for 7 days at 37 °C, and their DNA was extracted by the method previously described by Boom et al. [13]. For M. capricolum subsp. capripneumoniae, samples of DNA already extracted were provided by Dr. Frey and Dr. Falquet of the Université de Friborg (FR) and Dr. Thiacourt and Dr. Manso-Silvan of CIRAD-INRA (FR).

Statistical analysis

Descriptive statistics were demonstrated as percentages. The association between the presence of Mollicute microorganisms and the sanitary variables and clinical signs were evaluated using the Chi-square test or Fisher exact test with a significance level of 5%, and the risk factor using odds ratio was also calculated [14].

Results

The characteristics of the studied ranching properties, origin of the animals, and individual characteristics are described at Table 2. In the case of property characteristics, the number of animals was 115 from 10 properties, which reflects a lack of data collected regarding properties 2 and 6. Properties were mainly commercial, meat producers that adopted an intensive production system. Most of the animals were females, and the most frequent clinical signs were altered pre-scapular and inguinal lymph nodes (22% and 13% respectively) and hyperemic genital and ocular mucosa (10% in both cases).

Table 2.

Frequency of individual characteristics of ranch properties and animals studied

		Number of animals	(%)
Ranch property variables
Type of ranch	Meat	75	65.2
	Milk	40	34.8
Purpose	Commercial	98	85.2
	Subsistence	17	14.8
Type of ranching	Intensive	105	91.3
	Extensive	10	8.7
Individual variables
Sex	Male	28	21.2
	Female	104	78.8
Pre-scapular lymph node	Altered	30	22.7
	Normal	102	77.3
Inguinal lymph node	Altered	18	13.6
	Normal	114	86.4
Submandibular lymph node	Altered	9	6.8
	Normal	123	93.2
Pulmonary auscultation	Altered	3	2.3
	Normal	129	97.7
Genital mucosa	Hyperemic	13	9.8
	Normal	119	90.2
Ocular mucosa	Hyperemic	13	9.8
	Normal	119	90.2

Regarding PCR results, Mollicutes were identified in 67%, 21%, and 7% (values rounded) in the nasal, genital, and ocular samples, respectively. Regarding species-specific PCR, only M. agalactiae (4% in nasal samples) and M. conjunctivae (1.5% in ocular samples) were detected (Table 3).

Table 3.

PCR results by sample

Samples	PCR	Number of animals
		Positive (%)	Negative (%)
Nasal	Mollicutes	89 (67.4)	43 (34.8)
	Ma	6 (4.5)	126 (95.5)
	Mc	0 (0.0)	132 (100.0)
	Mmc	0 (0.0)	132 (100.0)
	Mcc	0 (0.0)	132 (100.0)
	Mccp	0 (0.0)	132 (100.0)
Genital	Mollicutes	26 (20.8)	99 (79.2)
	Ma	0 (0.0)	132 (100.0)
	Mc	0 (0.0)	132 (100.0)
	Mmc	0 (0.0)	132 (100.0)
	Mcc	0 (0.0)	132 (100.0)
	Mccp	0 (0.0)	132 (100.0)
Ocular	Mollicutes	9 (6.8)	123 (93.2)
	Ma	0 (0.0)	132 (100.0)
	Mc	2 (1.5)	130 (98.5)
	Mmc	0 (0.0)	132 (100.0)
	Mcc	0 (0.0)	132 (100.0)
	Mccp	0 (0.0)	132 (100.0)

Correlations between the results of the PCRs, qPCR, and the variables analyzed are presented in Table 4. A statistical association was found between animals from intensive production sites and the presence of Mollicutes in nasal samples (OR, 6.417; CI, 1.551–26.546; p = 0.013). An association was also found between dairy farms and the presence of Mollicutes in ocular samples (OR, 6.441; CI, 1.235–33.581; p = 0.037), as well as between animals involved in extensive breeding and positive results of Mollicutes in genital samples (OR, 3.900; CI, 1.028–14.796; p = 0.035). Due to the low detection of M. agalactiae and M. conjunctivae, no statistical correlation was found between the results of the molecular analysis and the variables studied.

Table 4.

Correlation between PCR and qPCR and analyzed variables

Variables		MC NS	OR	IC	p Value	MC OS	OR	IC	p Value	MC GS	OR	IC	p Value	Ma NS	OR	IC	p Value	Mc OS	OR	IC	p Value
		+				+				+				+				+
Pulmonary auscultation	Normal	87	0.966	0.085–10.951	1.000	9	-	-	-	25	1.940	0.169–22.266	1.000	6	-	-	-	2	-	-	-
	Altered	2				0				1				0				0
Genital mucosa	Normal	89	1.097	0.318–3.784	1.000	9	-	-	-	23	1.161	0.295–4.565	1.000	6	-	-	-	2	-	-	-
	Hyperemic	80				0				3				0				0
Ocular mucosa	Normal	9	0.751	0.230–2.448	0.869	9	-	-	-	25	0.320	0.039–2.600	0.456	5	1.900	0.205–17.633	1.000	2	1.900	0.205–17.633	1.000
	Hyperemic	89				0				1				1				0
Pre-scapular lymph node	Normal	81	0.957	0.403–2.272	0.920	8	0.405	0.049–3.376	0.653	19	1.289	0.480–3.462	0.613	3	3.667	0.700–19.206	0.257	2	3.667	0.700–19.206	0.257
	Altered	8				1				7				3				0
Inguinal lymph node	Normal	89	0.425	0.155–1.164	0.090	9	-	-	-	24	0.432	0.093–2.014	0.435	6	-	-	-	2	-	-	-
	Altered	69				0				2				0				0
Submandibular lymph node	Normal	82	1.750	0.348–8.804	0.750	9	-	-	-	24	1.292	0.245–6.807	1.000	6	-	-	-	2	-	-	-
	Altered	7				0				2				0				0
Type of ranch	Cut	89	0.722	0.317–1.644	0.222	2	6.441	1.235–33.581	0.037	14	1.630	0.654–4.063	0.292	2	4.056	0.709–23.191	0.213	0	-	-	-
	Milk	54				6				11				4				1
Purpose	Subsistence	26	0.665	0.201–2.205	0.222	0	-	-	-	1	5.731	0.721–45.574	0.127	0	-	-	-	0	-	-	-
	Commercial	80				8				24				6				1
Type of ranching	Extensive	13	6.417	1.551–26.546	0.437	8	-	-	-	20	3.900	1.028–14.796	0.035	6	-	-	-	1	-	-	-
	Intensive	67				0				5				0				0

Discussion

To better understand the role of mycoplasmas in goat livestock in southwestern Bahia state, the present study aimed to identify these microorganisms using molecular tests. The main objective was achieved by detecting Mollicutes, two species of mycoplasmas in the studied population, and the association between the presence of these microorganisms and the characteristics of ranch properties.

In the intensive livestock production type with commercial purpose, Mollicutes were more frequently detected compared to other studies in the northeast region of Brazil, which often report on extensive production type [4, 15–18]. This result might be related to the fact that in the study area, ranches receive assistance from the State University of Southwest of Bahia (UESB). A transition to intensive production type ranching has been reported by Albuquerque et al. [18], and it is related to an interest in breeding exotic goats, implementing more modern ranching technology and improvements in the nutritional and sanitary management to maximize the profits.

In this study, most of the goats were healthy, without clinical signs or disorders; the most common clinical sign observed was congestion of nasal and ocular mucosa. However, Filgueira et al. [4] and Coelho et al. [16] detected high rates of diseases such as caseous lymphadenitis, mastitis, and keratoconjunctivitis in the animals they studied. The sanitary conditions of the herds studied probably can be related to the change to an intensive system, with a commercial character, in which sanitary and nutritional management were improved [19].

In the present study, Mollicutes were most detected in nasal and genital samples. The absence of clinical signs and the detection of these microorganisms is consistent with the knowledge that Mollicutes are opportunistic and are part of the normal microbiota of ruminants [20]. Despite this, we demonstrate that Mollicutes are present in goat herds in Bahia, and they were distributed differently among the clinical samples.

The detection of M. agalactiae in six nasal samples indicated a low percentage (4%) but showed the presence of this Mollicute in the studied region. Despite having been detected and reported for more than 75 years in Brazil [21], M. agalactiae remains an economic concern for Brazilian goat production, especially in the northeastern region, and they can still be found in several Brazilian states [22–26].

Similarly, low frequency of M. conjunctivae was observed. This species causes keratoconjunctivitis and blindness in goats [27–29], and its presence in Brazilian goat herds strongly suggests the need for intensification of the sanitary management. The first and unique isolation of M. conjunctivae in Brazil was described by Gregory et al. [30]. To the best of our knowledge, this is the second study that reports this microorganism in Brazilian goat herds and the first identified my molecular techniques.

The results obtained are consistent with Câmara, Silva, and Guerra [6], who note the failure in Mollicute detection, which can be related to the lack of standardization in the procedures to detect these bacteria in clinical samples [5, 31].

Mollicutes were detected more in nasal swabs in goats from intensive production sites than extensive production types (OR, 6.417; CI, 1.551–26.546; p = 0.013). Therefore, this study indicated that the closer proximity between animals as found in the intensive production type can significantly increase the risk of Mollicutes, since these microorganisms are mostly transmitted by direct contact [32–35]. In addition, an association between dairy farms and the presence of Mollicutes in ocular samples (OR, 6.441; CI, 1.235–33.581; p = 0.037) was detected, but this finding requires further studies to better understand this relationship.

We also detected an association between extensive production type and the presence of Mollicutes in genital samples (OR, 3.900; CI, 1.028–14.796; p = 0.035). This result might be related to the decreased reproductive health management in this type of production, facilitating the dissemination of these microorganisms, as observed by Santos et al. [36] in the state of Paraíba. Coelho et al. [16] noted that the lack of good husbandry practices in smaller herds, mostly extensive type, with rustic conditions, favoring the occurrence of the diseases that interfere with high-quality goat production [31].

Conclusion

It important to understand that there is an enormous need for research to provide epidemiological data with a focus on goat breeding in Brazil, which is even greater when considering the identification of mycoplasma species. Results such as those obtained in the present study with the identification of M. conjunctivae, a microorganism described only once in the literature from Brazil to present date, highlight the need for further epidemiological studies on these bacteria of relevance for goat breeding. We conclude from this study that mycoplasmas are present in goat herds in the southwestern region of Bahia, which reinforces the need for more focused studies in identifying mycoplasmas as well as for elaborating a health profile for this type of breeding in northeastern Brazil. For all these reasons, we consider our findings essential for defining sanitary measures to be taken to provide healthier and more profitable herds, thus ensuring better living conditions for rural goat ranchers.

The present research demonstrated the presence of Mollicute microorganisms in Brazilian goat herds, particularly two important opportunistic bacteria, M. conjunctivae and M. agalactiae. To the best of our knowledge, this is the first time that M. conjunctivae was detected in Brazilian goats by molecular testing. These results will help veterinarians better understand the sanitary conditions in the southwestern region of Bahia state and reinforce the need to improve sanitary and hygiene management to provide healthier and more profitable herds, ensuring animal welfare and better living conditions for rural ranchers. This information should be used for the dissemination and awareness of breeders and authorities in order to emphasize the importance of the Mollicutes as well as the need for better conditions of management and intensification of animal health control, reducing losses and improving the quality of the herd in the region.

Funding

This study was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP 09/54825-5) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Code 001).

Declarations

Conflict of interest

The authors declare no competing interests.