Frequency and cyst density of Sarcocystis spp. in the myocardium of alpacas (Vicugna pacos) according to sex and age in a province of the Peruvian Andes

DOI: https://doi.org/10.21203/rs.3.rs-2011935/v1

Abstract

Sarcocystis is a threat to food security in the Peruvian Andes. The Sarcocystis species that forms microcysts in the myocardium of alpacas is S. masoni n. sp. (known as "S. lamacanis"). There is a lack of current studies estimating the prevalence of this parasite in alpacas, although this information is crucial for the control and prevention of sarcocystosis. The aim of this study was to determine the frequency and cyst density of Sarcocystis masoni n. sp. in the myocardium of alpacas in Huancavelica, a province of the Andean region of Peru. Myocardial samples for histological analysis were taken from 104 alpacas slaughtered in an official abattoir in the area. All alpacas (100%) presented this parasite. About 44% (95% CI: 34.5-53.5%), presented from 31 to 45 microcysts per mm2 of myocardial histological slide (cyst density). Cyst density was significantly higher (p<0.05) as the age of the animals increased. In addition, cyst density was significantly higher (p<0.05) in 4- and 5-years-old males compared to females of the same age. These results confirm that myocardial sarcocystosis is highly endemic in alpacas in Peru.

Introduction

In recent years, scientific community has shown a growing interest in South American camelids. Mainly because these animals have remarkable physiological and adaptive traits which are crucial versus scarcity conditions, such as those that extensive animal production may have to face in the future (semi-arid grasslands, erratic rainfall patterns, etc.) due to the climate change and the reduction of agricultural areas (Zarrin et al., 2020). Today, South American camelids are particularly important for the economy and food security of the Andean populations of Peru, since these animals can convert forage with limited nutritional potential into resources that are highly valued for their high quality, such as fiber and meat (Quispe et al., 2009). However, several diseases (Rodriguez et al., 2015, 2017; Lucas et al., 2016; Sicha et al., 2020) affect alpacas, causing mortality and reducing their productivity performance. Sarcocystis infections also reduces their productivity levels in alpacas (Chavez et al., 2008; Lucas, 2012; Saeed et al., 2018), particularly in adult animals (Castro et al.., 2004).

Sarcocystis is a parasite that causes sarcocystosis in humans and animals worldwide. Sarcocystis has a strict two-host life cycle, with herbivores or omnivores being the intermediate hosts and carnivores or omnivores being the definitive hosts. The alpaca acts as an intermediate host for two species of Sarcocystis, S. aucheniae and S. masoni n. sp. (known as "S. lamacanis"). S. aucheniae develops macrocysts in the skeletal muscles of alpacas and its main definitive host is the dog (Céspedes et al., 2013, Vilca et al., 2013, Zacarías et al., 2013). Sarcocystis macrocysts pose a serious problem for the marketing of South American camelid meat, because they cause carcass confiscation at the slaughterhouse (Lucas, 2012) and because the consumption of undercooked infected meat can cause food poisoning syndrome in humans (Lucas, 2013; Vilca et al., 2013; Céspedes et al., 2013).

S. masoni n. sp. produces microcysts in cardiac tissue (Lucas et al., 2019a) and skeletal muscle (Moré et al., 2016). The definitive host of this parasite is still unknown (Moré et al., 2016), although some studies have suggested that canids also play this role (Leguía et al., 1989; More et al., 2016; Leguía y Santiago, 2019). Although there are several prevalence studies of S. aucheniae, those showing the frequency of Sarcocystis in the myocardium of alpacas date from the 1960s (Guerrero et al., 1967) and other more recent studies have been obtained with a limited number of animals (López-Torres et al., 2015; Lucas et al., 2019a). 

Histology is frequently used to diagnose Sarcocystis in muscle tissues (Bucca et al., 2011; Kojouri et al., 2011; López-Torres et al., 2015) as it could provide information such as the level of adaptation of the parasite to its host. For example, Leguía et al. (1990) reported that infection of alpacas with microcysts of S. lamacanis produces hemorrhages associated with the presence of schizonts in the vascular endothelium. In cattle, the presence of degenerated cysts produces inflammatory reactions against the parasites (Beyazit et al., 2007). In addition, cyst density as a measure of infection intensity (Falcón et al., 2010; Bucca et al., 2011; Lopez-Torres et al., 2015) can provide valuable information when relating it to some risk factors. However, these studies are expensive and time-consuming. In this context, slaughterhouse surveys have been used as an economic strategy to estimate the burden and impact of neglected parasitic diseases in the endemic regions of the Peruvian Andes (Lucas et al. 2019b; Arias-Pacheco et al., 2020). Although slaughterhouse records are not representative of the actual infection status, they are useful as a basis for estimating the real rate of infection and the impact of the disease, and are commonly used during the implementation of specific control programs (Alton et al., 2015; Carroll et al., 2017). Therefore, the objective of this study was to determine the frequency and cyst density of Sarcocystis in the myocardium of alpacas slaughtered in a Peruvian Andean province.

Methods

Study Site

The present study was conducted in one of the official South American camelid slaughterhouses (authorized by the Peruvian Veterinary Service – Ministry of Agriculture) in Huancavelica province (Fig. 1) (altitude 3680 m, latitude 12°78′S, longitude 74°98′W), one of the main alpaca-producing regions in the Andes of Peru (MINAGRI, 2015). In this region, the annual maximum and minimum temperatures are 15°C and 3°C, respectively. Huancavelica climate is cold, rainy and with two distinct seasons: a rainy season from November to April, and a dry season from May to October.

Animals and sample collection

The study was conducted in March 2019 at the municipal slaughterhouse of Huancavelica. A total of 104 alpacas were slaughtered during this period. The age of animals was determined by the observation of teeth eruption, according to Altamirano (1987), and ranged from 1 to 5 years.

Samples were taken from all slaughtered alpacas. Sections (1.5 cm × 1.5 cm) of heart apex were sampled at the slaughterhouse using a scalpel. Samples were stored in plastic bottles with 10% buffered formaldehyde and transferred to the Pathology Laboratory of the National Agricultural Health Service (SENASA) for further analysis.

Laboratory analysis

All tissue samples were routinely processed, dehydrated, and embedded in paraffin. Paraffin-embedded tissues were later sectioned into 7-μm-thick sections, and hematoxylin and eosin (H&E) staining was performed. Five slides stained with H&E were obtained for each sample. Sarcocystis microcysts were identified using an optical microscope (Primo Star, Carl Zeiss, Germany), with digital camera (AxionCam ERc5s, Carl Zeiss, Germany) and ZEN 2012 SP1 measurement software (Blue edition, Carl Zeiss). Microcysts have been described as bodies of 23-75 μm x 28-280 μm wide by length, showing a reddish fibrous membrane, without septa and with violet bradyzoites inside by H&E staining (Lucas, 2012; Lucas et al., 2019a). 

Counts of Sarcocystis microcysts were performed at 4X magnification on two representative areas of 25 mm2 per slide (10 counts per sample). The average of the counts was called cyst density and was expressed as microcysts/mm2 (media ± standard deviation).

Statistical analysis

Statistical analysis was conducted using SPSS v.25.0 (IBM Corp., Armonk, NY, USA). The prevalence of Sarcocystis and confidence intervals were obtained. The data was categorized based on the sex and age of the animals. To determine statistical differences among the groups with respect to cyst density, data normality was confirmed by the Shapiro-Wilk test and compared by a one-way ANOVA with a Tukey post-hoc test. P value was set in 0.05.

Discussions And Results

In this study, all alpaca hearts presented Sarcocystis microcysts. Other authors also found 100% prevalence rates of myocardium of alpacas infected with S. masoni n. sp. "lamacanis" (Guerrero et al., 1967; López-Torres et al., 2015; Lucas et al., 2019a). No inflammatory reaction against the microcysts was observed (Fig. 2).

Lucas (2013) previously suggested the reasons for the high prevalence of Sarcocystis in alpacas in Peru. The main factors contributing to the perpetuation of the parasite in the Peruvian Andes are the following: the lack of education on sarcocystosis control and prevention, repeated reinfection of the definitive host, which excretes a large number of oocysts daily and over a long period of time, and the oocysts’ resistant, as they are expelled to the environment sporulated and infective (Lucas, 2013).

This high prevalence may pose a public health problem, since Sarcocystis microcysts in alpacas contain a cyst-derived neurotoxin called sarcocystin. Sarcocystin can cause gastroenteric symptoms and respiratory problems in humans if infected meat is consumed undercooked (Leguía, 2003; Lucas, 2012; Saeed et al., 2018). In this regard, several domestic physical and chemical treatments have been used to sanitize alpaca meat infected with macrocysts of S. aucheniae (Céspedes et al., 2013; Vilca et al., 2013); however, there are no studies on the sanitation of meat infected with microcysts of Sarcocystis masoni n. sp. (“lamacanis”).

One of the limitations of this study is the lack of a precise place of origin of the animals. Owners seldom declare the exact location of the alpacas when an animal is brought to slaughter. As a result, all animals are considered to be from the same district as the slaughterhouse.

Fig. 3 shows that 44% (95% CI: 34.5-53.5%) of the animals had between 31 and 45 microcysts per mm2 of histological slide of the alpaca myocardium. Similar results were previously reported in Peru in alpaca (Lopez-Torres et al., 2015) and cattle (Falcón et al., 2010) myocardium. However, in Italy, Bucca et al. (2011) reported lower counts in cattle of ≤1 cysts per microscopic field of myocardium. 

These differences could be related to practices that are common in the Peruvian Andes, such as poor sanitary control during breeding and inadequate disposal of contaminated tissues after slaughter.

The number of microcysts/mm2 was higher in males than in females (66.6 ± 25.6 vs. 54.9 ± 18.9, respectively). In addition, the cyst density increases as the age of the animal increases (Table 1). Significant differences (p<0.05) were observed in the cyst density of 4- and 5-year-old males compared to females of the same age (Fig. 4). Moreover, the count of microcysts/mm2 was significantly higher (p<0.05) in alpacas older than three years, both in females and males, than in younger animals (Fig. 4). 

Cyst density provides an approximation of the animal's level of infection. Therefore, alpacas with higher levels of cyst density could have been infected with a higher parasite load. This could be a result of a longer exposure time to a permanently contaminated environment as reported for Sarcocystis aucheniae in llamas and alpacas (Velásquez et al., 2019). Furthermore, it could be indicative of a reduction in production performance of alpacas. In fact, Chavez et al. (2008) described that the higher the parasite load of infection with Sarcocystis lamacanis, the greater the loss on weight gain and hematocrit values of infected animals. 

Declarations

Author contribution: All authors contributed to the study conception and design. Material preparation, data collection, and laboratory analysis were performed by AR, JR and MQS. Data analysis was performed by JR and JRL. The first draft of the manuscript was written by JRL, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. 

Acknowledgements: We did not possess any financial source or support of any kind.

Data availability: Data will be made available on reasonable request

Conflict of Interest: The authors declare that they have no conflict of interest.

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Table

Table 1. Number of Sarcocystis sp. microcysts per mm2 of myocardial histological slides of alpacas from the province of Huancavelica, Peru, according to age and sex.

 

n

Media

SD

Sex

 

 

 

Male

55

66.6y

25.6

Female

49

54.9y

18.9

Age (years)

 

 

 

26

37.2c

3.8

2

21

41.1c

5.6

3

18

58.4b

7.6

4

20

75.3ab

12.3

5

19

92.2a

15.9

Total

104

60.8

23.3

a,b,c,y different letters in the same column indicate significant differences (p<0.05)