AAVS_14_1

Introduction

In the Philippines, goats are integral to rural livelihood, contributing to food security and income generation for smallholder farmers (Alcedo et al., 2015). Nueva Vizcaya, a key livestock-producing province in the northern part of the country, is home to thousands of goats raised primarily under backyard systems (Philippine Statistics Authority, 2023). Despite their importance, goat herds are vulnerable to diseases that can impair productivity, cause economic loss, and pose zoonotic risks.

One such disease is cryptosporidiosis, caused by the protozoan Cryptosporidium, which can cause diarrhea, especially in young animals, and can be fatal in severe cases (Delafosse et al., 2006; Noordeen et al., 2012). Infected goats may also serve as reservoirs of zoonotic genotypes (Noordeen et al., 2012). Challenges in disease control include the parasite’s resistance in the environment and the limitations of traditional diagnostic methods. Molecular techniques, particularly PCR, offer sensitive detection and allow species/genotype-level identification of Cryptosporidium spp. (Khurana and Chaudhary, 2018; O’Leary et al., 2021).

Although Cryptosporidium has been reported in humans (Cross et al., 1985; Labana, 2019), livestock (Laxer et al., 1988; Rivera and Yason, 2008; Villanueva et al., 2010; Labana et al., 2018), and water sources (Labana, 2019) in the Philippines, published data specific to goats remain limited (Rivera and Yason, 2008; Domingo et al., 2012; Labana et al., 2018). Nueva Vizcaya, a key goat-producing province within Region II (Cagayan Valley), is among the country’s major livestock-producing regions which has an active goat dispersal and loan program for backyard farmers. Goat production in the province is dominated by smallholder, backyard systems where protozoal diarrheal diseases may occur but remain underrecognized. Moreover, no published epidemiological data on Cryptosporidium in goats has been conducted in Nueva Vizcaya, and the level of awareness among goat farmers regarding this zoonotic pathogen is unknown.

Given these gaps, this study aimed to determine the presence of Cryptosporidium infection in goat kids in Nueva Vizcaya using microscopy and PCR, and to assess the Knowledge, Attitudes, and Practices (KAP) of goat farmers regarding this zoonotic parasite.

Materials and Methods

Study area and population

The study was conducted in Nueva Vizcaya, Philippines (16.3301° N, 121.1710° E), which comprises 15 municipalities (Figure 1). It is part of the agriculturally rich Region 2 (Cagayan Valley) and hosts over 97,000 goats, mostly on smallholder farms (Philippine Statistics Authority, 2023). The province spans 3,904 km² and experiences average annual rainfall between 131.32–274.43 mm and temperatures of 25–29°C.

Fecal samples were collected from 158 goat kids aged ≤6 months across 10 randomly selected municipalities. The sample size was calculated using the closest regional goat prevalence estimate of 11.4% from Mi et al. (2014), with a 95% confidence level and ≤5% margin of error.

Goat farmers (n=60) directly handling goats and aged ≥18 years were also surveyed using structured questionnaires to assess KAP on cryptosporidiosis. This questionnaire served as a preliminary KAP assessment intended to provide baseline insights that would later inform the development of a fully validated KAP tool. The KAP survey sample size was determined based on the list of goat raisers provided by the Provincial Veterinary Services Office at the time of data collection. All farmers on this list were contacted, resulting in the final sample of 60 respondents. The sample size was also constrained by logistical feasibility during the COVID-19 lockdown, which limited the field mobility and access to more remote farms by the researchers.

Sampling and data collection

Fresh fecal samples (~5g per sample) were collected per rectum using sterile gloves and stored in labeled containers in an ice chest. Information about goat and farm management was gathered through face-to-face interviews with goat farmers. The structured questionnaire, translated into Filipino, included both open- and closed-ended questions covering: (1) goat-related information (breed, sex, fecal consistency); (2) farm characteristics (municipality, farm age, location, distance from the nearest residence or body of water, herd size, production type, and production system); (3) management practices (water and feed sources, type of flooring, presence of kidding and isolation pens, frequency of vitamin supplementation and deworming); and (4) farmer demographics (age, sex, education, marital status, household size, years of goat-raising experience, prior training for goat raising).

A separate printed questionnaire assessed KAP, with questions on the clinical signs, modes of transmission, diagnosis, and prevention. Responses were used to construct three index variables for knowledge, attitude, and practice scores following predefined scoring criteria. All management and farm data were entered into an electronic data collection tool (EpiCollect© v4.2.0, Centre for Genomic Pathogen Surveillance, UK), while geographic coordinates of each farm were recorded using GPS.

Sample processing, storage, and microscopic examination

Approximately 5 g of fecal pellets were soaked in distilled water until softened, then strained through a double layer of sterile gauze into a 10 ml test tube and centrifuged at 5000 rpm for 10 minutes. The supernatant was discarded, and the sediment was divided into two specimen containers one with 80% ethanol and one without, each labeled with its original sample code.

Oocysts were concentrated using Sheather’s sugar flotation. A mixture of sediment and flotation fluid was centrifuged at 2500 rpm for 5 minutes, after which the top 2 ml was transferred to another tube, washed once with distilled water by centrifugation at 2000×g for 15 minutes, and the final sediment resuspended in 1–2 ml of distilled water. Samples were recorded and stored at 4°C until analysis.

For microscopic examination, smears were prepared from the fecal suspension and stained using the Modified Ziehl-Neelsen technique. Briefly, air-dried smears were fixed in methanol for 30 seconds, stained with carbol fuchsin for 5 minutes, decolorized in acid-alcohol for 15 seconds, counterstained with methylene blue for 1 minute, rinsed, and air-dried. Slides were examined under high-dry (40×) and oil immersion (100×) objectives for the presence of oocysts.

PCR detection

DNA was extracted from ~200 μl of each fecal sediment using the E.Z.N.A.® Stool DNA Kit (Omega Bio-Tek, USA) with minor protocol modifications. DNA concentration and purity were measured spectrophotometrically, and extracts were stored at −20°C until use. Positive controls (Cryptosporidium DNA) were obtained from the Parasitology Research Laboratory, Institute of Biological Sciences, College of Arts and Sciences, University of the Philippines Los Baños (Paller et al., 2024), while ultrapure nuclease-free water served as the negative control.

Nested PCR targeting the 18S SSU rRNA gene was performed using two primer sets. The first, described by Yusof et al. (2017), amplified ~655 bp in the primary reaction using primers N-DIAG-F2 (CAA TTG GAG GGC AAG TCT GGT GCC AGC) and N-DIAG-R2 (CCT TCC TAT GTC TGG ACC TGG TGA GT), and ~435 bp in the secondary reaction using primers CPB-DIAG-F (AAG CTC GTA GTT GGA TTT CTG) and CPB-DIAG-R (TAA GGT GCT GAA GGA GTA AGG). Each 10 μl reaction contained 5 μl GoTaq® G2 Colorless Master Mix (Promega, USA), 0.5 μl of each primer, 2 μl of DNA template (primary) or first-round product (secondary), and nuclease-free water to volume. Thermocycling for the primary PCR consisted of initial denaturation at 95°C for 5 min; 35 cycles of 94°C for 45 s, 68°C for 1 min, and 72°C for 1 min; followed by a final extension at 72°C for 10 min. The secondary PCR used the same composition with 40 cycles of 94°C for 45 s, 60°C for 1 min, and 72°C for 30 s.

Products were resolved by electrophoresis on 2% agarose gels stained with ethidium bromide and visualized under UV illumination (Quantum, Vilber, Germany) alongside a 100-bp DNA ladder. Amplicons of the expected 435 bp were considered presumptive positives. Five such products were excised, purified using the NucleoSpin® Gel and PCR Clean-up Kit (Macherey-Nagel, Germany), and sequenced by Macrogen (Seoul, South Korea). Sequence identity was assessed via BLAST (NCBI GenBank). None of the sequences matched Cryptosporidium reference sequences.

To confirm results, nested PCR was repeated using the primers of Xiao et al. (1999), which target a ~1325 bp product in the primary reaction (primers SSU-F2: 5′-TTC TAG AGC TAA TAC ATG CG-3′ and SSU-R2: 5′-CCC TAA TCC TTC GAA ACA GGA-3′) and ~825 bp in the secondary reaction (forward: 5′-GGA AGG GTT GTA TTT ATT AGA TAA AG-3′; reverse: 5′-AAG GAG TAA GGA ACA ACC TCC A-3′). No positive amplicons were obtained with the second primer set.

Statistical analysis for KAP

Knowledge (max score: 26), attitude (max: 8), and practice (max: 24) scores were constructed. Wilcoxon rank sum tests assessed demographic associations. Thresholds: ≥80% for adequate knowledge and ≥18 points for good practice.

Results and Discussion

Detection of Cryptosporidium spp. in goat kids

Microscopic examination of all 158 fecal samples using the Modified Ziehl-Neelsen technique revealed no detectable Cryptosporidium oocysts. Meanwhile, nested PCR targeting the 18S SSU rRNA gene (Yusof et al., 2017) generated amplicons of approximately 435 bp in five samples. However, sequencing and BLAST analysis showed that none of these amplicons matched Cryptosporidium reference sequences. A second nested PCR using the primers of Xiao et al. (1999) was performed, but no positive amplicons were produced, confirming the absence of Cryptosporidium DNA in all tested samples. Similar reports of non-detection of Cryptosporidium in goats have been documented in Poland (Majewska et al., 2000), Tunisia (Soltane et al., 2007), and Mongolia (Burenbaatar et al., 2008).

Non-detection in other studies has been attributed to low environmental burdens (Majewska et al., 2000; Soltane et al., 2007). In the present study, most farms had small herd sizes (mean: 6 goats/farm). Although daily manure removal and smaller herds may help reduce fecal accumulation (Majewska et al., 2000), these practices should not be taken as indicators of comprehensive hygiene. Several important management gaps including the absence of isolation pens (96%) and reliance on untreated water sources (68%) were evident and could support the transmission of enteric pathogens in theory. Therefore, the absence of Cryptosporidium cannot be explained solely by effective hygiene.

The lack of detection could have been due to temporal and biological factors. Sample collection was done during the hot, dry months of April and May, when Cryptosporidium detection in tropical regions is often lowest and higher occurrence typically reported during cooler or rainy periods (Santín, 2013). Moreover, intermittent and low-intensity oocyst shedding could reduce the likelihood of detecting infections at very low levels. These considerations highlight the importance of year-round surveillance and sampling across multiple seasons to more accurately determine true prevalence and capture potential temporal variation. This finding should also be interpreted considering the study’s sample size, which was sufficient for detecting moderate prevalence, but may not have been sufficient to identify very low-level infections (Burenbaatar et al., 2008).

An additional factor that may be relevant to disease dynamics in the province is the active goat dispersal and loan program. Such programs may reduce the risk of disease introduction when animals are sourced from well-managed farms. On the other hand, without screening for protozoan pathogens such as Cryptosporidium, infected animals from a common supplier could be distributed simultaneously across multiple backyard farms, inadvertently facilitating pathogen spread. However, the specific health-screening procedures applied to dispersal animals were not disclosed during the study The influence of the dispersal program on the present findings is therefore uncertain and underscores the need for transparent, standardized health-screening protocols in livestock distribution initiatives.

Risk factors for Cryptosporidium spp. infections

Animal- and farm-level characteristics among the sampled population are presented in Tables 1 and 2. The 158 goat kids sampled across 25 farms were primarily raised for meat production. They were predominantly of mixed Anglo-Nubian × Philippine native breed. Most farms were located in rural areas, had small herd sizes, and used tethering as the primary production system. Farm management practices varied, with many farms lacking vitamin supplementation, deworming programs, and isolation facilities for sick animals. Contact with other domestic animals was common, with fowls and sheep being the most frequent.

Table 1: Animal-level characteristics of goat kids (n = 158) sampled from 25 farms in Nueva Vizcaya.

Variable	Category	n	%
Sex	Male	88	55.7
Sex	Female	70	44.3
Breed	Philippine native	43	27.2
	Mixed breed	103	65.2
	Anglo-Nubian	5	3.2
	Boer	4	2.5
	French Alpine	3	1.9
Fecal consistency	Diarrheic	30	19.0
Fecal consistency	Non-diarrheic	128	81.0

Table 2: Farm-level characteristics of the 25 sampled goat farms in Nueva Vizcaya.

Farm age (years)	< 5	5	20.0
Farm age (years)	≥ 5	20	80.0
Farm location	Rural	23	92.0
Farm location	Urban	2	8.0
Distance from nearest residence (m)	< 100	22	88.0
Distance from nearest residence (m)	≥ 100	3	12.0
Distance from nearest body of water (m)	< 100	13	52.0
Distance from nearest body of water (m)	≥ 100	12	48.0
Herd size (heads)	< 10	15	60.0
	11–20	5	20.0
	> 21	5	20.0
Production type	Meat-type	25 farms	100.0
Production system	Tethering	12	48.0
	Extensive	4	16.0
	Semi-extensive	5	20.0
	Intensive (zero-grazing)	4	16.0
Water source	Untreated	17	68.0
Water source	Treated	8	32.0
Feed source	Pasture alone	20	80.0
	Commercial + pasture	5	20.0
Type of flooring/ground	Soil/grass	19	76.0
	Slatted (elevated)	5	20.0
	Paved	1	4.0
	Table continues on next page............
Farm age (years)	< 5	5	20.0
Kidding pen	Present	4	16.0
Kidding pen	Absent	21	84.0
Isolation pen for sick animals	Present	1	4.0
Isolation pen for sick animals	Absent	24	96.0
Vitamin supplementation frequency (months)	None	17	68.0
	1	4	16.0
	2	1	4.0
	3	1	4.0
	> 4	2	8.0
Deworming frequency (months)	None	17	68.0
	1	6	24.0
	> 4	2	8.0
Farm perimeter	Fenced	15	60.0
Farm perimeter	Non-fenced	10	40.0
Goats in contact with other domestic animals (non-exclusive categories)	None	128	81.0
	Fowls	10	6.3
	Cats	4	2.5
	Dogs	11	7.0
	Sheep	2	1.3
	Pigs	2	1.3
	Cattle	1	0.6

Majority of the diarrheic goat kids were sampled in the adjacent municipalities of Dupax del Norte and Dupax del Sur (46.7%), suggesting a possible localized concentration of enteric disease in this area. The presence of diarrhea in 30 kids across 11 farms suggests ongoing circulation of other enteric pathogens. Because this study focused specifically on Cryptosporidium, testing for bacterial, viral, or other parasitic agents was not undertaken. This is an important limitation, as pathogens such as Escherichia coli, rotavirus, and Eimeria spp. are common causes of diarrhea in young goats (Khurana and Chaudhary, 2018). Future studies should incorporate diagnostic testing for these agents using microscopy, fecal flotation, bacterial culture or PCR, and viral antigen or molecular assays to provide a more comprehensive assessment of enteric disease burden.

Several management-related risk factors including untreated water sources (68%), soil flooring (76%), the absence of isolation pens (96%), and lack of consistent deworming and vitamin supplementation programs could facilitate the transmission of enteric pathogens (Thamsborg et al., 1990). Notably, none of the farms quarantined newly acquired animals, a critical biosecurity gap that could allow for the introduction of infectious agents. However, the presence of these factors does not necessarily translate into detectable infection at a single time point. Small herd sizes, seasonal timing of sample collection and the intermittent shedding patterns of Cryptosporidium may together result in parasite levels below the detection limits of microscopy or PCR. These considerations further support the need for expanded sampling efforts across different seasons and more geographical areas to more conclusively assess prevalence.

Knowledge, attitudes, and practices (KAP) of goat farmers

Knowledge of zoonotic diseases

Despite the absence of specific knowledge about Cryptosporidium, most goat farmers (95%) recognized that pathogens in goat feces can affect both goats and humans. However, the inability to name specific zoonotic pathogens may indicate a general lack of awareness of zoonotic infections such as cryptosporidiosis. Knowledge about modes of disease transmission was relatively high, particularly for fecal-oral routes. These findings are similar to KAP studies conducted among livestock owners in rural settings, where general awareness is high, but specific knowledge about zoonoses remains low (Singh et al., 2019).

The mean knowledge score was moderate (20.59/26), with 67% of respondents having adequate scores. Interestingly, goat farmers with less than 4 years of experience had significantly higher knowledge scores, possibly reflecting more recent exposure to training programs or extension services (Table 3). However, formal education level did not significantly impact knowledge scores, highlighting the importance of targeted farmer education regardless of background.

Table 3: Comparison of knowledge scores based on demographics of respondents using Wilcoxon rank sum test.

Variable	Category	Mean	n	p-value
Age (yrs)	< 48	20.72	30	0.84
Age (yrs)	≥ 48	20.43	28	0.84
Sex	Male	20.78	57	0.23
Sex	Female	17.00	3	0.23
Highest educational level attained	Did not attend college	20.63	14	0.40
Highest educational level attained	Attended college	20.51	43	0.40
Years in goat raising	< 4	21.24	24	0.01 *
Years in goat raising	≥ 4	20.16	36	0.01 *

* Significant at = 0.05

It should be acknowledged that the KAP findings may have been influenced by methodological limitations related to the questionnaire development process. Although the instrument was translated into Filipino to improve respondent comprehension, it was not subjected to back-translation, and no formal pilot testing was conducted prior to data collection. These limitations may have affected the clarity and interpretability of certain items. To strengthen the robustness of future KAP assessments, the incorporation of standardized translation protocols, including back-translation and formal pilot testing is recommended to ensure linguistic accuracy and conceptual equivalence. Therefore, the KAP component of this study should be regarded as a preliminary assessment, providing foundational insights to guide the development and future validation of a standardized KAP instrument.

Sources of information

Veterinarians and animal health workers were the primary sources of information for all respondents. However, only 10% of respondents reported receiving educational materials or protective items for disease prevention. This highlights a possible gap in the dissemination of educational and practical tools needed for improved biosecurity and zoonoses prevention. It also underscores the need for sustained extension and community-based training approaches to reinforce awareness and disease prevention practices.

Attitudes toward disease transmission

Attitude scores were generally high (mean: 7.85/8), and 93% of respondents demonstrated adequate attitudes. Younger goat farmers (<48 years) had significantly more favorable attitudes, which may reflect a greater openness to newer practices and information (Table 4). Almost all respondents acknowledged the potential of sick goats to transmit diseases to other goats and humans, and recognized economic losses due to illness and death. These attitudes, if paired with the right practices, are encouraging for potential uptake of preventive measures.

Table 4: Comparison of attitude scores based on demographics of respondents using Wilcoxon rank sum test.

Variable	Category	Mean	n	p-value
Age (yrs)	< 48	8	30	0.03*
Age (yrs)	≥ 48	7.68	28	0.03*
Sex	Male	7.84	57	0.64
Sex	Female	8	3	0.64
Highest educational level attained	Did not attend college	7.64	14	0.21
Highest educational level attained	Attended college	7.91	43	0.21
Years in goat raising	< 4	8	24	0.09
Years in goat raising	≥ 4	7.75	36	0.09

* Significant at = 0.05

Preventive practices

Most respondents reported sound practices: changing water daily, removing soiled feed and manure, isolating sick animals, and consulting veterinarians. The overall mean practice score was 20.8/24, with 82% of respondents classified as having adequate practices. However, only 43% of farmers submitted fecal samples for diagnosis, which may reflect economic or logistical constraints, or lack of awareness regarding the benefits of proper diagnosis. The practice of isolating sick goats (97%) was encouraging, although the lack of physical isolation facilities on nearly all farms may limit the effectiveness of such measures.

Table 5: Comparison of practice scores based on demographics of respondents using Wilcoxon rank sum test.

Variable	Category	Mean	n	p-value
Age (yrs)	< 48	20.70	30	0.58
Age (yrs)	≥ 48	20.96	28	0.58
Sex	Male	20.86	57	0.33
Sex	Female	19.67	3	0.33
Highest educational level attained	Did not attend college	21.29	14	0.12
Highest educational level attained	Attended college	20.56	43	0.12
Years in goat raising	< 4	21.46	24	0.02 *
Years in goat raising	≥ 4	20.36	36	0.02 *

* Significant at = 0.05

Interestingly, goat farmers with less than 4 years of experience had significantly higher practice scores (Table 5). Although farmers with less than four years of experience showed significantly higher knowledge and practice scores, the reasons for this association cannot be determined from the available data. The study did not collect information on training history, information-seeking behavior, or access to extension services that could clarify why newer farmers performed better in these domains. Further research is needed to identify the factors underlying this pattern, including whether newer farmers differ in exposure to training programs, access to information, or motivation to adopt recommended practices. Nonetheless, extension programs should continue to engage both new and long-standing farmers to ensure broad and equitable access to recommended management practices.

Conclusions

This study presents the first molecular and microscopic investigation of Cryptosporidium spp. in goat kids in Nueva Vizcaya, Philippines, alongside a preliminary KAP assessment among goat farmers. Although Cryptosporidium was not detected in any of the samples tested, the KAP results revealed substantial gaps in disease-specific knowledge, and several management practices observed across farms may facilitate the transmission of enteric pathogens more broadly. These findings highlight underlying vulnerabilities in the animal–human–environment interface that extend beyond Cryptosporidium itself.

The results point to systemic needs in farmer education, routine surveillance, and farm-level biosecurity. A One Health approach is therefore warranted because the study identified cross-cutting factors such as limited awareness of zoonotic risks, infrequent diagnostic submissions, and environmental and management conditions conducive to diarrheal disease transmission that require coordinated attention from multiple sectors.

A practical One Health framework for Nueva Vizcaya would involve collaboration among the animal health sector (municipal and provincial veterinarians, livestock technicians, and extension workers), the human health sector (rural health units), and the environmental sector (municipal environment and natural resources offices and sanitation services). Strengthening routine surveillance of animals and water sources, improving farmer training on manure management and zoonotic disease prevention, and establishing clearer pathways for diagnostic submissions would help reduce the risk of enteric infections in both livestock and communities. Future studies that integrate farm management data with individual KAP scores will enable more precise targeting of interventions and further support One Health implementation in the province.

Acknowledgments

The authors would like to thank Dr. Mylyn S. Liban and the Provincial Veterinary Services Office of Nueva Vizcaya for their support during sample collection. The authors also acknowledge Dr. Billy P. Divina, Dr. Kristina Andrea S. De Ramos, and the Parasitology Research Laboratory of the Institute of Biological Sciences, College of Arts and Sciences, University of the Philippines Los Baños, for their assistance during sample processing. The authors would also like to thank Ms. Dannah Zemira G. Junio and Mr. Paulo T. Abiera for their assistance during data analysis. Furthermore, the authors acknowledge the Capacity Development for Early Career Researchers (CareeR) Mentoring Program of the National Research Council of the Philippines (NRCP) for its support during the writing of this manuscript.

Novelty Statement

This study provides the first locally reported molecular and microscopic evaluation of Cryptosporidium spp. in goat kids in Nueva Vizcaya, Philippines, and is, to our knowledge, the first to integrate these techniques with a knowledge, attitudes, and practices (KAP) assessment among backyard goat farmers in the country. The results of the study provide baseline data to inform future farmer education and zoonotic disease prevention strategies.

Authors Contribution

Conceptualization- ICBP and RLG; Methodology- ICBP and RLG; Formal analysis- ICBP, RLG, CT, and PD; Writing (Original Draft)- ICBP: Writing (Review & Editing)- ICBP, RLG, CT, and PD; and Supervision- RLG, CT, and PD.

Ethical approval

Animal sampling was approved by the Institutional Animal Care and Use Committee of the University of the Philippines Los Baños (Protocol CVM-2022-11). The KAP survey was approved by the Institutional Review Board of the Institute of Tropical Medicine Antwerp (Ref. 1556/22). All participants signed informed consent forms, in line with ethical guidelines and the Declaration of Helsinki.

Generative AI and AI-assisted technology statement

The authors confirm that no generative AI were used in the analysis or interpretation of the scientific content of this manuscript. All content has been reviewed and approved by the authors.

Conflict of interest

The authors have declared no conflict of interest in the conduct and publication of this study.

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