1
UNIVERSIDADE FEDERAL DA PARAÍBA
CENTRO DE CIÊNCIAS AGRÁRIAS
INVESTIGAÇÃO EPIDEMIOLÓGICA DE INFECÇÕES DO
ÚBERE EM CABRAS PRIMÍPARAS E MULTÍPARAS NO PRÉ
E PÓS-PARTO POR UMA ABORDAGEM MOLECULAR
Iácome Sueliton Coelho Jácome
Médico Veterinário
2013
2
UNIVERSIDADE FEDERAL DA PARAÍBA
CENTRO DE CIÊNCIAS AGRÁRIAS
Investigação epidemiológica de infecções do úbere em cabras primíparas e
multíparas no pré e pós-parto por uma abordagem molecular
Iácome Sueliton Coelho Jácome
Orientador: Prof. Dr. Celso José Bruno de Oliveira
Coorientador: Prof. Dr. Danilo Tancler Stipp
Coorientador: Prof. Dr. Rafael Felipe da Costa Vieira
Dissertação apresentada ao Programa de PósGraduação em Ciência Animal do Centro de
Ciências Agrárias da Universidade Federal da
Paraíba, como parte das exigências para a
obtenção do título de Mestre em Ciência
Animal.
2013
3
UNIVERSIDADE FEDERAL DA PARAÍBA
CENTRO DE CIÊNCIAS AGRÁRIAS
IÁCOME SUELITON COELHO JÁCOME
Dissertação apresentada ao Programa de Pós-Graduação em Ciência Animal do
Centro de Ciências Agrárias da Universidade Federal da Paraíba, como parte das
exigências para a obtenção do título de Mestre em Ciência Animal.
APROVADO EM 12/12/2013.
BANCA EXAMINADORA
__________________________________________
Profº. Drº. Celso José Bruno de Oliveira
(Orientador)
__________________________________________
Profª. Drª. Maria das Graças Xavier de Carvalho
(Examinador)
__________________________________________
Profº. Drº. Suedney de Lima Silva
(Examinador)
4
DADOS CURRICULARES DO AUTOR
IÁCOME SUELITON COELHO JÁCOME – Nascido em Campina Grande, Paraíba,
ao dia 01 de junho de 1987. Concluiu o ensino médio em 2004, no Colégio Alfredo
Dantas. No mesmo ano concluiu o curso Técnico em Agropecuária na Escola
Agrícola Assis Chateaubriand, Campus Lagoa Seca da Universidade Estadual da
Paraíba. Em 2005, iniciou o curso de Medicina Veterinária pela Universidade Federal
de Campina Grande, concluindo em 2009. Durante esse período, foi PIVIC,
desenvolvendo atividades nas áreas de nutrição de pequenos ruminantes e
alternativas de convivência com semiárido. Ainda, foi monitor das disciplinas
Histologia e Embriologia Veterinária, Parasitologia Veterinária, Toxicologia, Clinica e
Patologia de Animais de Produção. Desde 2007, é Extensionista Rural da EMATER
Paraíba, desenvolvendo atividades de assistência técnica e extensão rural para
agricultores familiares dos Territórios do Médio Sertão e Borborema.
5
DEDICO:
A Deus, por ter iluminado minha vida desde sempre, em cada momento,
em cada ato, em cada fazer. Obrigado senhor por mais uma graça alcançada.
Aos meus pais, Pedro e Sueli Jácome, por todo o apoio, carinho e
confiança, sem os quais essa conquista não seria possível.
Amo vocês.
6
AGRADECIMENTOS
À minha avó (Maria Inácia), pelo apoio e carinho. Apesar de seus 89 anos,
apresenta-se mais saudável que eu. Valeu vó.
Aos meus irmãos (Adriana, Giácome, Yanglio, Pedro, Niácome e Rosalina),
principalmente os dois últimos, pelo carinho, amizade, momentos de harmonia e
felicidade que dividimos durante todo esse tempo.
Aos amigos e irmãos (Ticiano, Syduane, Jefferson e Alisson) pelo incentivo e
empenho, tornando essa conquista possível.
À minha esposa (Débora Duarte), a mulher dos olhos mais lindos que possam
existir, que me conquistou pelo seu apoio, carinho, compreensão e amor recíprocos
e que me incentiva a ir mais além. Obrigado. Te amo.
Aos colegas da U.O. de Lagoa Seca (Venancio, Juarez, Chico, Nereida, Maria
José e Salete) pela compreensão e respeito a mim dedicados.
Ao Coordenador regional de Campina Grande e o Presidente da EMATER
Paraíba (Sales Junior e Geovane Medeiros), pelo apoio e disposição, que tanto
contribuíram para essa conquista.
Aos amigos e colegas (Candice, Geovania, Camila, Angélica, Andreia,
Silvana, Mauro, Jéssica, Iago, Wellington, Alexandre, Heraldo e Denis Spricigo),
parceiros que adquiri durante a caminhada, que conviveram comigo em todos os
momentos, sendo de felicidade ou não, estavam sempre com a mesma dedicação e
carinho e me ajudaram a vencer muitos obstáculos.
À minha turma (2012), por tudo que vivemos juntos, momentos de amizade,
divertimentos que irão ficar na memória para sempre.
7
Aos funcionários do CCA (Claudia, Cleidson, Juliana e Boi), que estiveram
sempre à disposição quando precisei. Obrigado!
Aos professores da Zootecnia do Centro de Ciências Agrárias da UFPB,
principalmente (Dr. Paulo Sergio e Dr.ª Patrícia Givisiez), pelo privilégio do convívio,
atenção e apoio.
Ao meu orientador (Dr. Celso), e co-orientadores (Dr. Rafael Vieira e Dr.
Danilo Stipp) pelo apoio, dedicação, consideração e amizade durante minha
formação, pelos conhecimentos imprescindíveis que me passaram, extremamente
importantes para meu crescimento profissional. Obrigado por Tudo.
A Doutora Graça Xavier e o Doutor Suedney Silva pela colaboração e
participação na minha banca de defesa.
A todos os professores da PPGCAn do Centro de Ciências Agrárias da UFPB,
pelos conhecimentos que me passaram, indispensáveis para o sucesso profissional.
Enfim, a todos que contribuíram de forma direta e indireta para a
concretização dessa vitória, que não é só minha, mas sim de todos nós.
viii
SUMÁRIO
Página
RESUMO GERAL............................................................................................
11
ABSTRACT......................................................................................................
13
CONSIDERAÇÕES GERAIS...........................................................................
15
CAPÍTULO
I
-
OCCURRENCE
AND
EPIDEMIOLOGICAL
INVESTIGATION OF PRE AND POSTPARTUM UDDER INFECTIONS IN
PRIMIPAROUS AND MULTIPAROUS GOATS BY A MOLECULAR
APPROACH
17
Abstract…………………………………………………………...…………………
19
1. Introduction…...………………………………………………………...……….
21
2. Materials and Methods………………………………………………………...
22
2.1. Study design and samplings……………………………………………..
22
2.2. Microbiological isolating and identification…………………………...
23
2.3. Somatic cell counts………………………………………………………..
23
2.4. Genotyping by Rep-PCR………………………………………………….
23
3. Results and Discussion.………………………………………………………
24
4. Conclusion...…………………………………………………………………….
29
5. References…………………………………………………………………...…..
33
Referencias Bibliográficas…………………………………………………...…..
36
ix
LISTA DE TABELAS
Página
Table 1. Frequency of Staphylococcus in different sample sources
taken from two small-scale goat milk production systems in
Paraiba, Northeastern Brazil………………………………….…...
30
x
LISTA DE FIGURAS
Página
Figure 1. Dendogram illustrating the genotypic relatedness of
staphylococci
(n=50) isolated from different sample
sources in a small-scale goat milk production system
(Farm A) in Paraiba State, Northeastern Brazil…….…………
31
Figure 2. Dendogram illustrating the genotypic relatedness of
staphylococci
(n=67) isolated from different sample
sources in a small-scale goat milk production system
(Farm B) in Paraiba State, Northeastern Brazil.......................
32
xi
INVESTIGAÇÃO EPIDEMIOLÓGICA DE INFECÇÕES DO ÚBERE EM CABRAS
PRIMÍPARAS E MULTÍPARAS NO PRÉ E PÓS-PARTO POR UMA ABORDAGEM
MOLECULAR
RESUMO GERAL - A mastite continua sendo a doença infecciosa mais importante
economicamente nas espécies leiteiras. No entanto, o conhecimento sobre a
epidemiologia da infecção do úbere em caprinos é escasso, principalmente nas
regiões em desenvolvimento. O objetivo deste estudo foi reunir informações sobre a
epidemiologia da mastite em cabras primíparas e multíparas criadas em sistemas de
produção familiar na Paraíba, Nordeste do Brasil, o maior produtor de leite de cabra
da América Latina. Um estudo longitudinal composto por três amostragens (uma préparto e duas pós-parto) foi realizado em duas propriedades de caprinos de leite.
Foram coletadas amostras de secreção de leite pré-parto e leite pós-parto de cabras
primíparas e multíparas, como também swabs a partir da superfície de tetas, interior
das narinas, mãos dos ordenhadores, brete, baia e ambiente de sala de ordenha. A
secreção de leite, leite e swabs foram coletados para análise de cultura
microbiológica. Além disso, a contagem de células somáticas e Califórnia Mastite
teste (CMT) foram realizados nas amostras de leite. Os isolados selecionados nas
propriedades A (n=50) e B (n=67) foram genotipados por Rep- PCR. Mastite
subclínica em cabras primíparas no pré-parto causada por estafilococos foi
detectada em ambas as fazendas, embora associada a diferentes espécies. A
análise de genotipagem indicaram infecção persistente por Staphylococcus (S.)
aureus em um animal desde o pré-parto, repetindo-se nas amostragens seguintes,
possuindo os isolados grande parentesco clonal. Entretanto, o mesmo não foi
observado em uma infecção por S. haemolyticus, devido a diferentes padrões
genotípicos serem observados por bactérias isoladas de pré e pós-parto da mesma
metade do úbere. Uma fraca correlação foi observada entre os métodos de
diagnóstico da mastite. Embora estafilococos genotípicamente relacionados e
clones, tenham sido identificados a partir de várias fontes, incluindo o meio ambiente
xii
e as superfícies do corpo de cabras primíparas e multíparas em lactação, isolados
de secreção de leite e leite de cabras primíparas apresentaram padrões genotípicos
diferentes, sugerindo que a mastite subclínica em cabras primíparas no pré-parto
pode ter vias de transmissão específicas a serem descobertas.
Palavras-chave:
Staphylococcus
epidemiologia
molecular,
mastite
caprina,
Rep-PCR,
xiii
EPIDEMIOLOGICAL INVESTIGATION OF PRE AND POSTPARTUM UDDER
INFECTIONS IN PRIMIPAROUS AND MULTIPAROUS GOATS BY A
MOLECULAR APPROACH
ABSTRACT - Mastitis continues to be the most important infectious disease in
milking species in terms of economical burden. However, knowledge on the
epidemiology of udder infections in goats is scarce, mainly in developing regions.
The aim of this study was to gather information about the epidemiology of mastitis in
primiparous and multiparous goat does reared in family production systems in
Paraiba, Northeastern Brazil, the leading goat milk producer in Latin America. A
longitudinal study comprised by three samplings (one prepartum and two
postpartum) was performed in two goat farms. Prepartum colostrum and postpartum
milk were sampled from primiparous and multiparous goats, and also swabs were
taken from the surface of teats, nostrils, milkers' hands, restraint devices and milking
room environment. Colostrum, milk and swabs were collected for culture analysis.
Besides, somatic cell counts and California mastitis test (CMT) were performed in
milk samples. Selected isolates in farm A (n= 50) and B (n=67) were genotyped by
Rep-PCR. Subclinical mastitis in prepartum primiparous goats caused by
staphylococci was detected in both farms, although associated with different species.
Genotyping analysis indicated persistent infection by Staphylococcus (S.) aureus in
one animal since the isolate obtained from prepartum colostrum was clonally related
to the isolate obtained in following samplings. However, the same was not observed
in an infection by S. haemolyticus because different genotypic patterns were
observed for bacteria isolated pre- and postpartum from the same half udder. A poor
correlation was observed amongst the mastitis diagnostic methods. Although
genotypic-related and even clonally-related staphylococci were identified from
various sources, including the environment and body surfaces of primiparous and
multiparous lactating goats, isolates from colostrum and milk from primiparous goats
xiv
showed different patterns, suggesting that prepartum subclinical mastitis in
primiparous goats might have specific and still unraveled transmission routes.
Keywords:
caprine
Staphylococcus
mastitis,
molecular
epidemiology,
Rep-PCR,
15
CONSIDERAÇÕES GERAIS
O leite é considerado o alimento mais completo que existe na natureza,
principalmente quando observada sua constituição e importância para os mamíferos.
Nos últimos anos, o consumo de leite caprino vem aumentando em decorrência de
suas vantagens nutricionais quando comparadas ao leite bovino. Aliando-se a essa
nova oportunidade, a cadeia produtiva do setor tem desenvolvido inúmeras
tecnologias para o beneficiamento do leite em derivados, agregando valor aos
subprodutos tornando competitiva a atividade diante da exigência mercadológica
atual.
O Nordeste concentra a maior parte do rebanho caprino leiteiro do Brasil,
sendo uma das atividades mais importantes do Semi-árido. No Estado da Paraíba, a
atividade é praticada principalmente pela agricultura familiar, tendo significante
expressão sócio-econômica, sendo composta por uma grande variedade de
propriedades com diferentes características e formas de manejo, topografia,
condições edafo-climáticas e de produção, muitas vezes limitando a eficiência
produtiva e reprodutiva dos rebanhos.
O Cariri Paraibano, região tradicional e maior produtora de leite caprino,
apresenta um arranjo produtivo local em desenvolvimento, favorecendo a
organização da produção entre os produtores, proporcionando acesso a diversos
canais de comercialização, sejam eles institucionais e/ou livres. Entretanto, apesar
de todo o envolvimento da cadeia produtiva da atividade na economia da região,
ainda há uma precariedade nos sistemas de produção, comprometendo a nutrição e
a sanidade dos rebanhos.
A diminuição da produção e produtividade por perdas de animais associadas
a enfermidades são comuns. Uma das mais importantes e descritas é a mastite,
acometendo fêmeas caprinas de diversas raças, apresentando-se de várias formas
entre animais e rebanhos. Do ponto de vista sanitário, a prevenção da mastite deve
ocorrer antes mesmo do parto nas cabritas, havendo, dessa forma, preocupação
para que elas cheguem à produção em estado sanitário adequado, desenvolvendo
seu máximo potencial produtivo.
16
Como prevenção para essa enfermidade, estudos voltados para os aspectos
epidemiológicos são bastante relevantes, sendo o ponto de partida para a adoção de
medidas sanitárias dentro da unidade de produção. Os estudos epidemiológicos têm
avançado muito nos últimos anos. Hoje é possível fazer relações com maior
precisão, necessária para determinar causas de surtos de doenças em populações
aplicando-se ferramentas moleculares, norteando medidas de controle imediatas e
eficazes.
Os métodos moleculares de genotipagem vêm sendo cada vez mais
aplicados em estudos epidemiológicos, auxiliando na investigação sobre fontes de
contaminação e meios de transmissão, possibilitando o monitoramento e/ou o
registro dos canais pelos quais os agentes acometem diferentes células e tecidos.
Até o presente, no Brasil e no mundo, não há relatos da ocorrência de mastite
em cabritas no período pré-parto. Vários estudos têm demonstrado o envolvimento
de diversos agentes na etiologia das mastites em cabras e recentemente em
novilhas de vacas proporcionando a identificação de vários agentes patogênicos e
diferentes fontes e vias de infecção sendo o agente mais freqüentemente
identificado pertencente ao gênero Staphylococcus (HAVERI et al., 2008; DE
VLIEGHER et al., 2012; CASTELANI et al., 2013).
Diante da importância da caprinocultura leiteira para o Semi-árido e não
sendo do nosso conhecimento relatos sobre mastite em cabritas na região nordeste,
esse estudo tem por objetivo investigar a ocorrência e as fontes e vias de infecção
mamária em cabritas no período pré-parto criadas em sistema de produção familiar
no Estado da Paraíba, utilizando-se de ferramentas moleculares de genotipagem
para determinação dos aspectos epidemiológicos da infecção.
17
CAPITULO I
EPIDEMIOLOGICAL INVESTIGATION OF PRE AND POSTPARTUM UDDER
INFECTIONS IN PRIMIPAROUS AND MULTIPAROUS GOATS BY A
MOLECULAR APPROACH
18
Epidemiological investigation of pre and postpartum udder infections in primiparous
and multiparous goats by a molecular approach
I. S. C. Jácome1, F. G. C. Sousa2, C. M. G. de Leon2, P. E. N. Givisiez2, D. T. Stipp1, R. F.
V. Costa1, W. A. Gebreyes3,4, and C. J. B. Oliveira*2,4
1
Department of Veterinary Sciences, Federal University of Paraiba, Brazil, Areia-PB, 58397-
000
2
Department of Animal Science, Federal University of Paraiba, Brazil, Areia-PB, 58397-000
3
Department of Veterinary Preventive Medicine, College of Veterinary Medicine, The Ohio
State University, USA, Columbus-OH 43210
4
Veterinary Public Health and Biotechnology Global Consortium (VPH-Biotec), The Ohio
State University, USA, Columbus-OH 43210
*
Corresponding author:
Celso J. B. Oliveira
Centro de Ciências Agrárias – UFPB
Departamento de Zootecnia
Rod. PB079, km 12
Areia-PB, Brazil
58397-000
Phone: +55 83 3362 2300 ext.3259
Fax: +55 83 3362 2504
E-mail: [email protected]
19
ABSTRACT
Mastitis continues to be the most important infectious disease in milking species in terms of
economical burden. However, knowledge on the epidemiology of udder infections in goats is
scarce, mainly in developing regions. The aim of this study was to gather information about
the epidemiology of mastitis in primiparous and multiparous goat does reared in family
production systems in Paraiba, Northeastern Brazil, the leading goat milk producer in Latin
America. A longitudinal study comprised by three samplings (one prepartum and two
postpartum) was performed in two goat farms. Prepartum colostrum and postpartum milk
were sampled from primiparous and multiparous goats, and also swabs were taken from the
surface of teats, nostrils, milkers' hands, restraint devices and milking room environment.
Colostrum, milk and swabs were collected for culture analysis. Besides, somatic cell counts
and California mastitis test (CMT) were performed in milk samples. Selected isolates in farm
A (n= 50) and B (n=67) were genotyped by Rep-PCR. Subclinical mastitis in prepartum
primiparous goats caused by staphylococci was detected in both farms, although associated
with different species. Genotyping analysis indicated persistent infection by Staphylococcus
(S.) aureus in one animal since the isolate obtained from prepartum colostrum was clonally
related to the isolate obtained in following samplings. However, the same was not observed in
an infection by S. haemolyticus because different genotypic patterns were observed for
bacteria isolated pre- and postpartum from the same half udder. A poor correlation was
observed amongst the mastitis diagnostic methods. Although genotypic-related and even
clonally-related staphylococci were identified from various sources, including the
environment and body surfaces of primiparous and multiparous lactating goats, isolates from
colostrum and milk from primiparous goats showed different patterns, suggesting that
20
prepartum subclinical mastitis in primiparous goats might have specific and still unraveled
transmission routes.
Keywords:
caprine
mastitis,
molecular
epidemiology,
Rep-PCR,
Staphylococcus
21
1. INTRODUCTION
Around 91% of the Brazilian goat herds are located in Northeast region of Brazil (IBGE,
2006) and Paraiba State is currently the leading goat milk producer in Latin America.
Although the region shows great potential to consolidate as an important economic sector, the
production system is comprised mainly by family producers owning small dairy goat herds.
The milk yielded in those farms is pasteurized in small scale dairy plants, and then bought by
the Government to be distributed to public schools in the scope of social programs, such as
"Fome Zero" [No Hunger]. Therefore, the developing goat milk production chain still have to
overcome basic problems that compromise nutrition and health status of the herds.
Although information on mastitis in the region is scarce, previous reports indicate that
coagulase-negative staphylococci (CNS) are the main pathogens associated with mastitis in
lactating goats (Peixoto et al., 2012). Besides, studies have shown staphylococcal
contamination in goat milk produced in Northeastern Brazil (Oliveira et al., 2011, Cavalcante
et al., 2013), indicating that udder infections are important sanitary problems for the region.
In the past few years, studies demonstrating mastitis by Staphylococcus in replacement heifers
have raised intriguing questions about the epidemiology of such infection in dairy herds, since
the pathogen has been considered a contagious agent basically transmitted among lactating
animals during milking practices (Castelani et al., 2013).
Recent findings suggesting
environment as a potential source of Staphylococcus causing mastitis in cattle (Anderson et
al., 2012) clearly demonstrate that detailed knowledge on the epidemiology of udder
infections might contribute significantly to the establishment of control measures in order to
reduce infections.
The aim of this study was to investigate the occurrence of udder infections by Staphylococcus
in replacement goats and to gain information on the epidemiology of mastitis in primiparous
22
and multiparous goats in two goat production units in Paraiba State. To our knowledge, this is
the first report describing the occurrence of mastitis in prepartum primiparous goats and the
use of a molecular approach to investigate possible transmission routes of udder infections in
milking goats.
2. MATERIALS AND METHODS
2.1 Study design and samplings
A longitudinal study was carried out in two goat farms (A and B) located in the municipalities
of Areia and Bananeiras, Paraiba State, Brazil. From each farm 10 animals (7 primiparous and
3 multiparous lactating goats) were selected and identified. The number of sampled animals
was defined by convenience, based on the number of breed does in the herd. In each farm, the
animals and environment were sampled three times. The first sampling was performed before
labor and the two consecutive samplings performed at 30 and 60 days after first sampling.
The samples included prepartum colostrum (n=39) and postpartum milk (n=78) from
primiparous and multiparous goats. These samples were collected aseptically according to
National Mastitis Council procedures (NMC, 1996). Additionally,swabs were taken from goat
teats (n=60) and nostrils (n=60), milkers' hands (n=6), milking restraint devices (n=6), stalls
(n=6) and the milking room environment (n=6).
Before samplings, animals were examined physically and udder health was evaluated
clinically and by means of strip cup test for clinical mastitis detection and California Mastitis
test (CMT) for subclinical mastitis diagnosis, according to recommendations (NMC, 1996).
Positive samples were considered for reactions showing moderate (++) and strong (+++)
agglutination reactions.
23
2.2. Microbiological isolating and identification
Conventional microbiological culture by Staphylococcus was performed according to NMC
(1996). Shortly, samples were streaked onto blood agar, MacConkey and Mannitol salt agar
plates. After aerobic culture for 24 to 48 h at 35 °C, colonies were analyzed by Gram staining,
catalase and oxidase. Staphylococcus spp. isolates were tested for coagulase in tubes. Isolates
from milk and colostrum from primiparous and multiparous goats were identified by a
microplate biochemical panel (Combo PC33, Siemens Healthcare, Malvern, PA) using a
semi-automated system (Autoscan 4, Siemens Healthcare, Malvern, PA) in parallel to the
determination of the minimal inhibitory concentration (MIC) for the antimicrobial
susceptibility test.
2.3. Somatic cell counts
Somatic cell counts (SCC) in the milk samples was determined by direct microscopy
according to Prescott e Breed (1910). The slides were prepared in duplicates and stained with
pyronin-Y-based stain as described by Zeng (1997). Cells were counted using 60 microscopic
fields and calculations were performed using a factor considering the objective field of the
microscopy (Nikon, Eclipse E-200).
2.4. Genotyping by Rep-PCR
Genomic DNA was extracted using phenol:chloroform:isoamyl alcohol (25:24:1), as
described by Sambrook et al. (1989) and DNA concentrations were adjusted to 50 ng/µL.
Rep-PCR amplifications were performed using the RW3A primer (5'-TCG CTC AAA ACA
ACG ACA CC-3') according to van der Zee et al. (1999) in a 25-µL final volume containg
Taq DNA polimerase buffer (x1), MgCl2 (3 mM), dNTPs (200 µM each), primer RW3A (1
24
pmol), Taq DNA polimerase (1U) and DNA template (100ng). Amplification was performed
in a thermal cycler (Biometra, Germany) according to the following conditions:
initial
denaturation (3 min at 94°C), 30 amplification cycles (1 min at 94ºC, 1 min at 50ºC and 2
min at 72ºC), and a final extension for 5 min at 72ºC. Amplified products were separated by
electrophoresis in 2% agarose gel at 80V for 2 h. After staining with 0.01% GelRed (Biotium,
USA), gel images were acquired using a Kodak Gel Logic 100 Imaging System instrument
equipped with a UV transilluminator (Carestream Health Inc., USA). Gel was analyzed and
dendogram was built using Bionumerics software v. 7.1 (Applied Maths NV, Keistraat,
Belgium) using Dice similarity index and unweighted pair group average (UPGMA) cluster
analysis. Banding patterns were compared with 2% band position tolerance and 1%
optimization.
To evaluate the agreement between diagnostic tests CMT, SCC and microbiological culture of
the Kappa test was used. The discriminatory power of Rep-PCR was calculated by the index
(D value) proposed by to Hunter (1990).
3. RESULTS AND DISCUSSION
Clinical mastitis has been detected in a half udder of one prepartum primiparous goat (1/14)
from farm B in all three samplings. Staphylococcus (S.) aureus was isolated from the
colostrum and milk of this animal in the three samplings but a co-infection by S. hycus was
confirmed in the second sampling. On the other hand, none of the other animals (13
primiparous and 06 multiparous goats) investigated in the study showed clinical mastitis
throughout the study period.
The frequency of positive microbiological culture in the three samplings performed in farms
A and B is shown in Table 1. Bacteria were recovered from 19 of the 117 (16.2%) milk and
25
colostrum samples, from which 5 (8.3%) originated from farm A and 14 (24.5%) from farm
B. Staphylococcus genus predominated amongst the isolates and the majority of them were
CNS. Micrococcus genus was observed in two samples.
Amongst the 5 isolates from farm A, S. haemolyticus (n=2), and S. epidermidis (n=1) were
identified, besides Micrococcus (n=2). Staphylococci organisms were also isolated from teat
swabs (n=25), nostril swabs (n=26), milkers' hand swabs (n=2), milking restraint device
(n=3), stall (n=2) and wall of the milking room (n=3) in this farm throughout the study period.
S. haemolyticus was isolated from colostrum of a primiparous goat before the beginning of
lactation and, interestingly, S. epidermidis was isolated from the same half udder in the
second sampling. S. haemolyticus was also isolated from milk collected from a multiparous
goat in the third sampling. Micrococcus was isolated from milk secretion of a primiparous
goat before lactation and from a different primiparous goat in the third sampling.
In farm B, Staphylococci were isolated from four primiparous goat and one multiparous goat.
Identified Staphylococci species included S. aureus (n=5), S. hyicus (n=2), S. epidermidis
(n=1), S. auricularis (n=1), S. cohnii (n=1), S. hominis (n=1), S. xylosus (n=1). Positive
samples for Staphyloccoci were also identified in teat swabs (n=27), nostril swabs (n=26),
milkers' hand swabs (n=3), milking restraint device (n=3), stall (n=1) and milking room wall
(n=3).
The isolate recovered from one primiparous goat showing clinical mastitis in farm B was S.
aureus. This bacteria was also isolated from the same half udder in the two following
samplings. Conversely to what was observed in the infection by S. haemoliticus in Farm A,
the S. aureus isolates from the same udder in the prepartum and postpartum sampling periods
shared similar genotypic patterns, suggesting the persistence of S. aureus infection in the
udder (Figure 1).
26
Considering subclinical infections in farm B, one sample of prepartum colostrum from a
primiparous goat was infected and S. aureus was isolated from the left half udder in the first
and second postpartum samplings. Besides, S. hyicus and S. epidermidis were isolated from
the right half udder in the first and second samplings, respectively. S. cohnii was also isolated
from prepartum colostrum of a primiparous goat, but the infection did not persist since no
bacteria was isolated in the following samplings. S. xylosus was recovered from milk of a
primiparous goat in the third sampling. From a multiparous goat, S. auricularis and S. hominis
were isolated from the same half udder in the first and third samplings, respectively.
Subclinical mastitis caused by coagulase-positive staphylococci was found in the present
study and has also been previously reported in multiparous goats (Ali et al., 2010; Cavalcante
et al. 2013). Nevertheless, the majority of mastitis-associated staphylococci identified in the
present study were coagulase-negative, corroborating previous reports in Brazil (Peixoto et
al., 2013) and in other countries (Aulrich and Barth, 2008; Koop et al.; 2010, Gebrewahid et
al., 2012). The results of the present study corroborate the those findings that CNS might play
a key role as a mastitis pathogen in the goat species. However, the persistence of infections
over the lactation period was clearly observed in the S. aureus infections only (farm B), since
the genotypic patterns of S. haemolyticus isolated from prepartum colostrum of kid goats and
from multiparous goats (Figure 1) were not similar. The fact that a variety of CNS has been
associated to mastitis in goats and the differences in the etiology of mastitis in the only two
investigated farms indicate that the epidemiology of the staphylococcal mastitis in the goat
species is complex.
Considering the diagnostic methods for subclinical mastitis, 9 of the 20 (45%) goats were
positive by microbiological culture throughout the study. Considering the CMT results, 17
animals (7 from Farm A and 10 from farm B) were positive for mastitis. Out of the 78 teat
27
samples evaluated by CMT (40 from farm A and 38 from farm B), 28 (35.9%) were positive
and 50 (64.1%) showed no or weak agglutination, and then considered negative.
Somatic cell counts ranged from 1,305,066 to 16,361,082 cells/mL in primiparous goats and
from 509,294 to 13,623,625 cells/mL in multiparous goats, with mean values of 4,696,441
and 3,542,779 cells/mL, respectively. Mean values for somatic cells were higher in
primiparous goats compared to multiparous goats.
A poor agreement was observed amongst the methods to detect mastitis. The kappa
coefficient values between culture and CMT, culture and CCS and CMT and CCS were
considered poor (0.159, 0.062 and 0.159, respectively).
The genotypic relatedness of the isolates obtained in farms A and B is presented in the
dendograms shown in Figures 1 and 2, respectively. A total of 45 and 50 distinct genotypic
patterns by Rep-PCR were generated in farms A and B, respectively. The Rep-PCR method
was able to differentiate epidemiological related staphylococci of the same species. Besides,
the high D-values observed for farm A (0.99) and Farm B (0.98) isolates indicate the RepPCR as a useful tool in typing staphylococci organisms associated with goat farms. Indeed,
Rep-PCR using RW3A has been successfully used to discriminate epidemiologically related
Staphylococcus aureus of animal (van der Zee et al., 1999; Peixoto et al., 2013) and human
(Deplano et al., 2000) origins.
Staphylococci isolates from farm A were assigned to ten distinct clusters (A to J) and one
single isolate (K) from milkers' hand swab showing a distinct genotypic profile. The majority
of the isolates (n=11) were grouped in cluster B. Isolates from teat and nostril swabs obtained
from various primiparous and multiparous lactating goats showed similar profiles and some of
them showed identical fingerprints by Rep-PCR. However, no clonal relatedness could be
detected between isolates from colostrum taken from primiparous goats before labor and
28
isolates from other sources. For example, S. haemolyticus isolated from colostrum of a
primiparous goat before labor (cluster H) was not related to S. haemolyticus obtained from
milk and teat swabs of lactating animals (cluster B). Isolates from at least one environmental
sample was observed in each cluster. In some clusters, no difference could be detected in the
genotypes of staphylococci from primiparous and multiparous goats. Some isolates from
milkers' hand shared a similar genotypic profile with staphylococci from environment sources
but no relation to mastitis causing agents was observed.
In farm B (Figure 2), seven clusters (A to G) of staphylococci were identified in the
dendogram. As also observed in farm A, some staphylococci from environment showed nondistinguished patterns compared with isolates from nostril and teat swabs from kid goats and
goats as well as environment. In cluster D, for example, clonally related isolates originated
from those sources (nostril and teat swabs, and environment) were identified. The majority of
isolates obtained from colostrum and milk from goats before labor grouped in cluster C.
Interestingly, isolates from milk samples collected from primiparous goats after labor were
also grouped in this cluster, which did not include isolates from environmental sources and
milkers' hand swabs.
It´s noteworthy that some staphylococci isolated from swabs taken from primiparous goats
(nostril and teat) before labor and from the wall of the milking room were clonally related,
even so primiparous goats had no access to the milking room before lactation. A high
genotypic relatedness was observed between one S. auricularis isolated from kid goat milk
secretion and one isolated taken from the wall of the milking room (Figure 2, Cluster E). This
finding together with the similar genotypic patterns of isolates from milkers' hand swabs,
environment and animals indicate the spread of staphylococci organisms in dairy goat milk
production systems. Milkers could indeed play a role on the dissemination of staphylococci,
29
since isolates from primiparous goats teat swabs (non-lactating animals) were clonally related
to isolates from the hand of milkers (Figure 2, Cluster D).
This fact is linked to the
characteristics of the dairy goat production systems in Northeastern Brazil, where primiparous
and multiparous goats share the same environment in the prepartum period, facilitating the
spread of microorganisms. Recent reports demonstrated a higher risk for udder infections in
heifers raised in contact to multiparous cows and the same mastitis causing agents were
involved (De Vliegher et al., 2012; Castelani et al., 2013). On the other hand, despite the high
similarity amongst staphylococci from environmental sources and body surfaces of goats, the
genotyping results indicated that those organisms were not causal agents of mastits in
primiparous goats. Other sources or staphylococci reservoirs should be further investigated.
5. CONCLUSIONS
Udder infections in primiparous kid goats in the pre-partum period can occur and persist in
the lactation, especially if caused by the S. aureus species. Although staphylococci organisms
are spread in the environment and body surfaces of primiparous and multiparous goats, the
causative agents of prepartum mastitis in the former seems to not be genotypically related to
those agents and further research to identify the infection sources and routes of mastitis in
primiparous goats are warranted.
Acknowledgments
The authors are thankful to Banco do Nordeste (Fundeci/BNB) and National Council for
Scientific and Technological Development – CNPq (proc. 483103/2007-1) for financial
support.
30
Table 1. Frequency of Staphylococcus in different sample sources taken from two small-scale
goat milk production systems in Paraiba, Northeastern Brazil.
31
84.2
76.2
95.7
70.6
100
95
90
85
80
75
70
65
60
55
50
45
40
Figure 1. Dendogram illustrating the genotypic relatedness of staphylococci (n=50) isolated from different
sample sources in a small-scale goat milk production system (Farm A) in Paraiba State, Northeastern
Brazil.
Rep_PCRi
Rep_PCRi
Key
Source
Swab Sampling Genus
.
Goat
212
Teat
1ª
.
Milk
secretion kid goat .
1ª
A
91.7
90.9
88.3
69.0
.
Milker
2ª
.
Stall
3ª
.
Milk
kid goat
2ª
.
Milker
3ª
.
Containment
box
3ª
.
Goat
115
84.7
2ª
.
Milker
2ª
. goat 209
Kid
76.4
Nasal 1ª
1ª
.
Containment
box
90.0
.
Goat
212
B
Teat
. goat 205
Kid
Teat
C
80.0
73.2
87.5
79.8
D
62.7
94.1
56.5
90.6
E
82.6
F
48.9
82.4
77.0
G
Teat
1ª
. goat 219
Kid
Teat
2ª
.
Goat
195
Nasal 3ª
.
Milker
3ª
.
Containment
box
1ª
70.7
Teat
3ª
. goat 203
Kid
Teat
1ª
. goat 200
Kid
Teat
1ª
.
Stall
1ª
.
Containment
box
2ª
.
Milker
3ª
.
Room
Wall
1ª
.
Containment
box
2ª
Nasal 3ª
.
Room
Wall
2ª
.
Containment
box
3ª
1ª
.
Containment
box
H
.
Goat
115
58.8
Teat
1ª
.
Milk
secretion kid goat .
45.4
81.9
I
66.4
92.3
84.0
38.0
J
K
83.3
2ª
. goat 210
Kid
Teat
. goat 202
Kid
Nasal 2ª
.
Stall
3ª
.
Room
Wall
3ª
72.9
43.4
3ª
2ª
.
Room
Wall
55.2
. goat 205
Kid
Nasal 1ª
. goat 210
Kid
Nasal 1ª
.
Containment
box
3ª
. goat 210
Kid
Nasal 1ª
. goat 203
Kid
Nasal 1ª
.
Containment
box
. goat 200
Kid
.
Milker
S. haemolyticus
3ª
.
Goat
195
.
Goat
185
85.7
48.1
. goat 211
Kid
.
Room
Wall
78.5
S. haemolyticus
1ª
3ª
.
Milk
goat 115
66.6
2ª
3ª
.
Milk
goat 115
90.0
79.2
S. epidermidis
Nasal 3ª
.
Milker
81.4
72.3
S. haemolyticus
.
ATCC
25923
77.6
66.8
2ª
.
Milk
secretion kid goat .
3ª
Nasal 1ª
2ª
S. haemolyticus
32
66.7
100
95
90
85
80
75
70
65
60
55
50
45
40
35
30
25
Figure 2. Dendogram illustrating the genotypic relatedness of staphylococci (n=67) isolated from different
sample sources in a small-scale goat milk production system (Farm B) in Paraiba State, Northeastern
Brazil.
Rep_PCRi
Rep_PCRi
Key
Source
Swab
Sampling
. Goat 1003
kid
Teat
3ª
1ª
.
Milk
secretion kid Goat 004
59.3
A
90.9
51.4
72.7
B
92.3
.
Goat
713
Teat
2ª
. Goat 944
kid
Teat
3ª
. Goat 969
kid
Teat
3ª
.
Milk
kid Goat 969
3ª
.
Room
Wall
2ª
.
Goat
74
64.1
Teat
. Goat 1003
kid
87.0
S. cohnii-cohnii
S. epidermidis
3ª
3ª
.
Milk
Goat 713
Genus
Nasal
2ª
Teat
2ª
S. hominis-hominis
.
ATCC
25923
77.0
. Goat 983
kid
87.5
87.5
53.7
43.3
72.6
93.3
84.2
81.9
67.0
C
64.5
83.3
80.0
51.5
.
Milk
kid Goat 944
3ª
S. aureus
.
Milk
secretion kid Goat 969
1ª
S. aureus
. Goat 004
kid
Nasal
1ª
. Goat 944
kid
Teat
1ª
.
Milk
secretion kid Goat 969
1ª
S. aureus
.
Milk
kid Goat 944
2ª
S. aureus
.
Milk
secretion kid Goat 944
1ª
S. aureus
.
Milk
kid Goat 969
2ª
S. hyicus
.
Milk
secretion kid Goat 944
1ª
S. hyicus
.
Goat
713
Nasal
3ª
.
Goat
613
Teat
2ª
.
Goat
613
Nasal
2ª
3ª
.
Containment
box
1ª
.
Containment
box
72.0
39.7
92.3
86.0
61.3
80.6
55.8
D
75.0
. Goat 969
kid
Teat
1ª
. Goat 005
kid
Nasal
1ª
3ª
.
Milker
. Goat 983
kid
Nasal
3ª
.
Goat
93
Nasal
2ª
.
Goat
93
Teat
2ª
. Goat 944
kid
Teat
2ª
. Goat 983
kid
Teat
3ª
.
Goat
808
Nasal
3ª
.
Room
Wall
Nasal
1ª
. Goat 983
kid
Teat
1ª
3ª
.
Milk
kid Goat 1003
61.9
85.7
34.1
. Goat 944
kid
Nasal
1ª
. Goat 969
kid
Teat
2ª
1ª
.
Room
Wall
88.9
1ª
.
Milk
secretion Goat 713
E
’
59.6
93.8
31.5
87.5
. Goat 944
kid
Nasal
2ª
.
Goat
613
Nasal
3ª
. Goat 1003
kid
Nasal
3ª
. Goat 005
kid
Nasal
3ª
. Goat 944
kid
Nasal
3ª
.
Goat
93
Nasal
1ª
. Goat 1003
kid
Nasal
1ª
. Goat 004
kid
Teat
1ª
.
Goat
613
Nasal
1ª
75.7
2ª
.
Containment
box
F
’
93.3
61.8
88.9
. Goat 1003
kid
Teat
2ª
. Goat 983
kid
Nasal
2ª
3ª
.
Milker
24.8
85.7
.
Goat
713
Teat
1ª
.
Goat
93
Teat
1ª
1ª
.
Stall
.
Goat
713
73.3
Teat
49.7
.
Goat
74
Nasal
3ª
.
Goat
808
Teat
3ª
. Goat 005
kid
Teat
2ª
.
Goat
613
Teat
3ª
. Goat 1003
kid
Teat
1ª
40.9
80.0
71.1
3ª
3ª
.
Containment
box
G
’
.
Room
Wall
2ª
.
Containment
box
2ª
.
Milker
1ª
69.3
S. xylosus
S. auricularis
33
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