Assiut University web-site: www.aun.edu.eg

DETECTION OF NEOSPORA CANINUM AND COXIELLA BURNETII ANTIBODIES IN MILK AND SERUM OF INFECTED DAIRY CATTLE

GERGES, A.A. ¹; MONA, SOBHY ¹; FATHI, A.²; HAMS, M.A. MOHAMED  ³;

ABOU-GAZIA, KH.A. ¹ and MOHAMED, H.F. ¹

¹ Department of Reproductive Diseases, ARRI, ARC, Giza, Egypt

² Unit of Immunity, ARRI, ARC, Giza, Egypt.

³ Dept. of Microbiology, Fac. of Vet. Med. SVU, Qena, Egypt

Received: 21 May 2018;   Accepted: 25 June 2018

INTRODUCTION

The rate of reproductive problems in cattle have been increasing over years and causes many economic problems in the bovine industry and public health which represented a standpoint because of their zoonotic importance, a number of infectious agents for instance, bacteria, viral, fungus and parasitic are recognized to have a direct impact on reproductive health of cattle (Yoo, 2010).

Neosporosis, caused by the obligate intracellular parasite protozoan called Neospora caninum (N. caninum), it has a highly preference for cattle and dogs as hosts and induces a serious economic losses like abortion, temporary anestrus and less milk production in dairy (Bruhn et al., 2013 and Almería et al., 2017).

Q fever  is  a zoonotic  and  prevalent  disease in most

Corresponding author: Dr. HAMS, M.A. MOHAMED

E-mail address: hams.mohamed@vet.svu.edu.eg

Present address: Dept. of Microbiology, Fac. of Vet. Med. SVU, Qena, Egypt

countries of the world, it caused by a gram negative bacteria called Coxiella burnetii (C.burnetii). These bacteria has many hosts include mammals, birds and arthropods like ticks (Angelakis and Raoult 2010).

There are two antigenic forms of C. bumetii that are imperative for serologic diagnosis of Q fever are the phase I (S-LPS) is avirulent form in ticks with smooth lipopolysaccharide and phase II (R-LPS) is avirulent microorganism with rough LPS with whole-cell antigens (Peter et al., 1985).

The shedding of C.burnetii into the environment happens primarily by birth products, especially placenta, are heavily infected with C. burnetii also it could be gained from milk and other excreta of infected animals (Schmeer et al., 1987). Coxiella can persist in the environment for long periods and may carried for a long distances through the wind (Kirkan et al., 2008). In cattle, late abortion, mastitis and infertility are the major clinical manifestations related to coxiellosis (Arricau et al., 2003).

Serological methods represent the most widely recognized, broadly available and frequently used tools for the diagnosis of infectious diseases (Garcia-perez et al. 2011; An and Ghattof, 2018). These methods are renowned from all other diagnostic methods by providing indirect evidence of infection or exposure by detecting host antibodies reactive against infections.

Enzyme linked immune sorbent assay (ELISA) and immune fluorescence antibody (IFA) generally considered the main serological method for diagnosis the C.burnetii infection (Meekelenkamp et al., 2012 and Anati-Pirouz et al., 2015).

So this study aimed to describe the seroepidemiological situation of N. caninum and C. burnetii specific antibodies in milk and serum of dairy cattle by using ELISA and IFA in Upper Egypt Governorates.

MATERIALS AND METHODS

1- Samples collection:

A total of 200 milk and serum samples (100 for each) were collected according the international ethics for animal experiments from 100dairy cattle suffering from infertility and abortion during the period from January to December 2017 in upper Egypt governorates (Table1). Pooled milk and serum samples were collected from the same dairy cattle.

a-Serum samples

Blood samples (5ml) were collected from the jugular vein of each cow in vacuum tubes without anticoagulant, the serum was decanted according (Peter et al., 1988).

b-Milk samples

each milk sample were centrifuged at 1000rpm for 10 min, skimmed milk was collected in sterile screw capped bottle and stored at -20℃ until examination (Frossling et al., 2003).

NB. All samples were subjected to examination for Brucella infection by using Rose Bengal test (rabid slide agglutination test, ID. Vet Genetics, france) for detection of Brucella antibodies in serum and Milk Ring Test (hematoxylin-stained antigen manufactured by the State Biological Laboratory, Institute of Veterinary Preventive Medicine, Ranipet, India) for detection of Brucella antibodies in milk samples (Alton et al., 1988).

2- Detection of N.caninum antibodies in serum and milk samples by using ELISA test:

The serum and whey-milk samples were screened for detection of Neospora caninum antibodies (IgG) by commercially available diagnostic indirect ELISA kit in serum, or milk for multispecies (ID. Vet innovative Diagnstics Louis Pasteur. Grabeis, France) using X check software program. All control tests were performed in duplicate. The diluent, wash solution and dilution buffer were primed according to manufacture instruction. The optical density (OD) values of the wells were read with ELISA reader (Titertek Multiskan plus MK II), at a wave length of 450 nm. The attendance of antibody to N. caninum was resolute in samples according to (S/P) ratio for each sample. The positive serum samples have S/P ratio ≥ 50 while positive milk samples have S/P ratio ≥ 30, the incidence of seropositive animals in the governorates was compared using Chi-square test (Analytical Software Package, Statistix version 1.0, 1996).

3-Detection of C. burnetii antibodies in serum and milk samples by ELISA and IFA

A-Detection of C. burnetii IgG in serum and milk samples by using ELISA test:

The samples were tested for the existence of anti-C. burnetii antibodies (IGg) by ELISA test (ELISA Cox kit, LSI-Laboratories Service International, Lyon, France) according to Yang et al. (2015). The optical density (OD) values of the wells were read with ELISA reader (Titertek Multiskan plus MK II), at a wave length of 450 nm. The S/P ratio was categorized in three classes in serum samples: positive 50<S/P ≤80, doubtful 40<S/P≤50 and negatives S/P C. burnetii when S/P>40, doubtful 30< S/P≤ 40 and negative S/P≤30 according to manufacturer's instructions.

B- Detection of C.burnetii antibodies by indirect Immunofluorescence technique (IFA):

Milk and serum were tested for IgM and IgG antibodies against C. burnetii phase II by using slides coated with C. burnetii phaseII (VIRCELL,S.L.pza. slides, SPAIN).

1- Detection of IgM

-  serum samples were diluted (1:2) and treated with anti-human IgG sorbent. Different dilution of treated serum were prepared 1:24, 1:48, 1:96 and placed in wells with positive and negative control, after several washing a IgM FITC conjugate solution was added to the wells, The steps were completed according manufacture instructions. The slides were examined under ultraviolet light of fluorescence microscope the reaction is positive when the apple-green fluorescence of coco-bacillary morphology can be observed. The reaction is negative when no fluorescence can be observed.

2-Detection of IgG

All samples were screened for different dilutions1:32, 1:64 and 1:128 by adding phosphate buffer saline (PBS). 20μl of each dilution were added to slide wells with control after incubation and several washing, IgG-fluorescein isothiocyanate (FITC) immune-conjugate was added to the wells. The initial dilution of 1:32 was considered as a negative dilution. The IgG phase II titer >32 was considered an indicative for chronic infection). The slides were examined under ultraviolet light by fluorescence microscope. The reaction is positive when the apple-green fluorescence of coco-bacillary morphology can be observed. The reaction is negative when no fluorescence can be observed.

RESULTS

All serum and milk samples included in this study were tested for Brucella infection by using rose bengal test and milk ring test. The tested samples showed a negative results.

In the current study, the data illustrated in table (2) and fig (1) showed that the N.caninum antibodies (IgG-ELISA) were detected in 29% of serum samples and in 10% of milk samples. The incidence ranged from 28% to 36% in serum samples while it ranged from 8%to12% in milk samples collected from cows farms in four governorates of Upper Egypt.

According to results in table (3) and fig (2) chronic infection (IgG for C.burnetii phase II) was cleared in 34% of serum samples (IgG titer >50) and 17% of milk samples (IgG titer >40), the higher incidence in serum samples was noticed in El-Fayoum governorate while in milk samples, El-Fayoum and Giza were the higher than other governorates.

The results demonstrated in table (4) and fig (3&4) revealed that recent infection of C.burnetii (IgM-IFA)appeared in 11% of serum samples and 4%of milk samples while the past infection (IgG-IFA) was showed in19% of serum samples and 8% of milk samples.

The comparative analysis between the results of IgG- ELISA and these obtained by IFA showed that ELISA is more accurate than IFA in decreasing the false negative results in serum and milk samples (Table 5).

Results in table (6) cleared that both C.burnetii and N. caninum antibodies were detected in the same serum and milk samples with percentage 14% and 4% among dairy cattle in 4 governorates in Egypt.

Tables

Table 1: Number of blood and milk samples collected from dairy cattle in different Egyptian governorates.

Table 2: Incidence of N. caninum antibodies in serum and milk samples by using ELISA test.

Table 3: Incidence of C.burnetii antibodies in serum and milk samples by using ELISA test.

Table 4: positive samples for C.burnetii antibodies in milk and serum samples by using IFA test.

Table 5: Comparison between by IFA test and ELISA test indetection IgG of C.burnetii in serum and milk samples.

Table 6: Positive samples for both C. burnetii and N. caninum antibodies in the same serum and milk samples by using ELISA test.

Figures

Fig (1): Incidence of N.caninum in serum and milk samples collected from dairy cattle of Upper Egypt governorates by using ELISA

Fig (2): Incidence of C.burnetii in serum and milk samples collected from dairy cattle of Upper Egypt governorates by using ELISA

Fig (3): The green fluorescence of IgM to C.burnetii phase II under ultraviolet light at a magnification of 400x by fluorescence microscope

Fig (4): The apple-green fluorescence of IgG to C.burnetii phase II under ultraviolet light at a magnification of 400x by fluorescence microscope.

DISCUSSION

Coxiella burnetii and Neospora caninum are an important reasons of reproductive problems in cattle, they causes a various signs in animals like infertility, early embryonic death, lingering calving seasons, abortion and stillbirth (Anderson, 2007 and Adis et al., 2018).

The serological methods represented the most widely recognized, available and commonly used tools for the diagnosis of infectious diseases by detection specific antibodies (Garcia-Pérez et al., 2011), moreover, the presence or absence of these antibodies in the blood of the host allowing for distinguishing time dependent for acute and chronic infection in serological profile (Guatteo et al., 2011).

Our study demonstrated a different levels of antibodies for N.caninum and C.burnetii in serum and milk samples of upper Egypt governorates but no sample recorded a positivity for Brucella antibodies by rose bangal test and milk ring test, these results in accordance with Softic et al. (2018) who concluded that a high antibodies titer to C.burnetii and N. caninum was recorded but Brucella spp. didn’t represent a risk in their study.

In this study the incidence of N. caninum were recorded in 29% of serum samples collected from four governorates in Egypt. (Table 2), these results in accordance with Fávero et al. (2017).

The incidence of N. caninum antibodies (IgG) in serum samples were reported in Beni-Sweif (36%), Giza (28%), El-Fayoum (28%) and Menia (24%) respectively by using ELISA test (Table 2 and fig 1). Lower results were recorded with (Kuruca et al., 2013 and Gerges et al., 2017).

While the incidence of N.caninum were recorded in 10% of in milk samples (Table 2). Our results in nearly similar with Schares et al. (2003) while the highest rates of infection was recorded in milk (26.66%) by Pritchard (2001).

Our results showed a differences in incidence of N.caninum antibody in four governorates of upper Egypt, This difference can be imputed to management and therapeutic system, water source, nutrition In addition to contacts of cattle with dogs, (Slapeta et al., 2002).

 In recent researches, the epidemiological studies on C. burnetii depend on serological tools in diagnosis (Rousset et al., 2010), because isolation of this pathogen remains time-consuming, risky and requires restricted biosafety level 3 laboratories due to the zoonotic nature of the microorganism (Fournier et al., 1998 and Field et al., 2000).

ELISA is easy to perform and is adapted for automation, on other hand the detection of IgG give an accurate diagnosis in determination the past infection of C.burnetii (Cowley et al., 1992; Wegdam-Blans et al., 2012).

In our results, the positive antibody titer of C. burnetii was determined the chronic infection in 34% of serum sample and in 17% of milk samples by using ELISA test (Table 3 and fig.2), higher results were recorded by Hussien et al. (2012) and Dean et al. (2013) on other hand Gwida et al. (2014) recorded a lower incidence of C.burnetii in serum samples collected from upper Egypt farms ranged from 2.9 to 26.7%.

El-Fayoum was recorded a significant increase (P>0.05) for C.burnetii infection in serum and milk samples than other governorates (Table 3 and Fig. 2), these variation in infection rate may be back to several factors like ahost, weather  conditions and the type of soil, (Fayer and Reid, 1982), also Adesiyun et al. (1996) reported that epidemiological studies differ in their design, laboratory and statistical analyses, types and number of samples which explained the limitation of the resemblance between results.

The extracellular infectious form of C.burnetii shed in milk, urine, and feces. It was found in high concentration (109 ID50/g) in placental tissue and amniotic fluid (Plummer, 2018). Once a domestic ruminant is infected, C.burnetii can localize in mammary glands, supra-mammary lymph nodes, placenta, and uterus (Agerholm, 2013).

It was noticed in current study that the incidence of C.burnetii in milk samples lower than serum samples although the samples were collected from the same animals (Table 3 and Fig. 2) this may be due the shedding of C.burnetii in milk discontinuous and always occur subsequent to parturition or abortion (Schmeer et al., 1987, Van den Brom et al., 2012 and Agerholm, 2013).

The results illustrated in table (4) and fig(3&4) showed that the recent infection (IgM titre>1:24) was recorded in 11% of serum samples and in 4% of milk samples while chronic infection (IgG titer ≥1:64) was recorded in 19% of serum samples and in 8% of milk samples by using indirect immunofluorcent test (IFA), our results nearly similar to (Sobhy et al. 2016), higher results were obtained by Htwe et al. (1992) who detected antibodies to C. burnetii in 29.5% of milk samples collected from healthy cattle and 84.3% of cattle with reproductive diseases in Japan by using IFA.

Our study focus on detection of IgM and IgG titre to phase II C.burnetii antigens by IFA, because the titer of antibodies to this phase reached to high magnitude in short time and persisted for along period, our results supported by Uhaa et al. (1994) who compared with  IgM and IgG to phase I antigens and phase II antigens, he found that IgM and IgG titer to phase II C.burnetii antigens remains for long time circulating in blood of the host in contrary with antibodies to phase I antigens disappearing during the convalescent phases of the illness while antibodies to phase II antigen was observed in acute and chronic infection.

Peacock et al. (1983) found that the raising in IgM and IgG titer to phase II C.burnetii were enough to eliminate the false-positive reactions against related antigens. Also Huebner et al. (1949) found that animals may be responsible for maintenance the chain of infection. However, the ways of transmission still needed further investigation.

Our results showed that the positivity rates of IgG to C.burnetii in serum and milk samples were detected in 34% and 17% by using ELISA while they were 19% and 8% by using IFA respectively (Table 5). It was noticed that the sensitivity of ELISA was higher than IFA in detection of IgG to C.burnetii, these results were supported by many previous studies which reported that ELISA, it is critical test to eliminate a variety of false positive and negative results during detection of C.burnetii antibodies (Cowley et al., 1992, Uhaa et al., 1994 and Waritani et al., 2017).

In the present study, both antibodies of C. burnetii and N. caninum was declared in the same samples by ELISA test, with percentage 14% in serum samples and 4% in milk samples (Table 6). Our results supported by García-Ispierto et al. (2010) who concluded the ability of the sharing Coxiella and Neospora antibodies in the same samples. Softic et al. (2018) reported that C. burnetii and N. caninum are a frequent causes of decreased reproductive consequences and reduced the reproductive performance   in cattle worldwide also the higher exposure to C. burnetii and N. caninum in different countries were a highlights need for targeted control of infectious causes of reproductive disorders in dairy cattle of the studied areas.

CONCLUSION

EILSA is an important tool in diagnosis of N. caninum and C.burnetii infection, the elevated altitudes of exposure to C. burnetii and N. caninum in this study need a targeted control for these infectious causes in dairy cattle. So, raising awareness is needed for animal owners, veterinarians, physicians, authorities and control strategies must be applied to protect against infection.

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